Mesenchymal Stem Cells In Hydrogels Research Articles

COMPARATIVE STUDY ON THE EFFECT OF COLLAGEN AND FIBRIN HYDROGELS OF VARYING STIFFNESS ON MESENCHYMAL STEM CELL PARACRINE ACTIONS

Mesenchymal stem cells (MSC) have potent immunomodulatory and regenerative effects via soluble factors. One approach to improve stem cell-based therapies is encapsulation of MSC in hydrogels based on natural proteins such as collagen and fibrin, which play critical roles in bone healing. In this work, we comparatively studied the influence of collagen and fibrin hydrogels of varying stiffness on the paracrine interactions established by MSC with macrophages and osteoblasts.Type I collagen and fibrin hydrogels in a similar stiffness range loaded with MSC from donants were prepared by modifying the protein concentration. Viability and morphology of MSC in hydrogels as well as cell migration rate from the matrices were determined. Paracrine actions of MSC in hydrogels were evaluated in co-cultures with human macrophages from healthy blood donors or with osteoblasts from bone explants of patients with osteonecrosis of the femoral head.Lower matrix stiffness resulted in higher MSC viability and migration. Cell migration rate from collagen hydrogels was higher than from fibrin matrices. The secretion of the immunomodulatory factors interleukin-6 (IL-6) and prostaglandin E2 (PGE2) by MSC in both collagen and fibrin hydrogels increased with increasing matrix stiffness. Tumor necrosis factor-α (TNF-α) secretion by macrophages cultured on collagen hydrogels was lower than on fibrin matrices. Interestingly, higher collagen matrix stiffness resulted in lower secreted TNF-α while the trend was opposite on fibrin hydrogels. In all cases, TNF-α levels were lower when macrophages were cultured on hydrogels containing MSC than on empty gels, an effect partially mediated by PGE2. Finally, mineralization capacity of osteoblasts co-cultured with MSC in hydrogels increased with increasing matrix stiffness, although this effect was more notably for collagen hydrogels.Paracrine interactions established by MSC in hydrogels with macrophages and osteoblasts are regulated by matrix composition and stiffness.

VP1: Combined Local Delivery of Tacrolimus and Stem Cells in Fibrin Gel is a Viable Potential Treatment for Enhancing Peripheral Nerve Regeneration

INTRODUCTION: The immunosuppressive and neuroregenerative potential of tacrolimus (FK506) may overcome the rejection of stem cells to enhance nerve regeneration. The aim of this study was to determine the feasibility and effectiveness of combining local, sustained delivery of tacrolimus (FK506) with stem cells. MATERIALS AND METHODS: The drug release profile of local FK506 over the course of 35 days was evaluated in phosphate-buffered saline (PBS). FK506 was incorporated into fibrin gel in poly(lactic-co-glycolic) acid (PLGA) microspheres in concentrations of 0.01 ng/mL and 100 ng/mL and incubated in phosphate-buffered saline (PBS) at physiological temperature. Microspheres were prepared and characterized according to validated protocols by Tajdaran et al. (2015). Adipose derived mesenchymal stem cells (MSCs) were cultured in collected PBS to mimic systemic exposure representing released concentrations at day 7, 15 and 28 from 0.01 ng/mL and 100 ng/mL microspheres. MSCs were cultured in the following groups: (i) hydrogel only, (ii) PLGA empty microspheres in hydrogel, (iii) Hydrogel Infused with Stem cells and Tacrolimus (HIST) 0.01 ng/mL, and (iv) HIST 100 ng/mL. Staining against CD90 was conducted to confirm stem cell characterization. MSC viability was evaluated at 24h, 48h, 72h, and seven days using Live/Dead staining and MTS assays. RESULTS: Microspheres containing 0.01 ng/mL showed depletion of FK506 by 13 days. FK506 microspheres containing 100 ng/mL revealed a sustained release up to 35 days. An inflation point was observed at day 15 due to erosion of gel, resulting in an increase of FK506 release. CD90 staining confirmed stem cell characterization. MSCs were viable up to seven days and no significant differences in viability were found between groups. CONCLUSION: Encapsulation of 100 ng/mL FK506 microspheres and MSCs in hydrogel is feasible and has strong potential to enhance survival of transplanted cells, which may ultimately lead to improved nerve regeneration.

Negative pressure wound therapy improves bone regeneration by promoting osteogenic differentiation via the AMPK-ULK1-autophagy axis

ABSTRACT Deficient bone regeneration causes bone defects or nonunion in a substantial proportion of trauma patients that urges for novel therapies. To develop a reliable therapy, we investigated the effect of negative pressure wound therapy (NPWT) on bone regeneration in vivo in a rat calvarial defect model. Negative pressure (NP) treatment in vitro was mimicked to test its effect on osteoblast differentiation in rat mesenchymal stem cells (MSCs) and MC3T3-E1 cells. Transcriptomic analyses, pharmaceutical interventions, and shRNA knockdowns were conducted to explore the underlying mechanism and their clinical relevance was investigated in samples from patients with nonunion. The potential application of a combined therapy of MSCs in hydrogels with negative pressure was tested in the rat critical-size calvarial defect model. We found that NPWT promoted bone regeneration in vivo and NP treatment induced osteoblast differentiation in vitro. NP induced osteogenesis via activating macroautophagy/autophagy by AMPK-ULK1 signaling that was impaired in clinical samples from patients with nonunion. More importantly, the combined therapy involving MSCs in hydrogels with negative pressure significantly improved bone regeneration in rat critical-size calvarial defect model. Thus, our study identifies a novel AMPK-ULK1-autophagy axis by which negative pressure promotes osteoblast differentiation of MSCs and bone regeneration. NPWT treatment can potentially be adopted for therapy of bone defects. Abbreviations: ADP, adenosine diphosphate; AICAR/Aic, acadesine; ALP, alkaline phosphatase; ALPL, alkaline phosphatase, biomineralization associated; AMP, adenosine monophosphate; AMPK, AMP-activated protein kinase; ARS, alizarin red S staining; ATG7, autophagy related 7; ATP, adenosine triphosphate; BA1, bafilomycin A1; BGLAP/OCN, bone gamma-carboxyglutamate protein; BL, BL-918; BS, bone surface; BS/TV, bone surface per tissue volume; BV/TV, bone volume per tissue volume; C.C, compound C; CCN1, cellular communication network factor 1; COL1A1, collagen type I alpha 1 chain; COL4A3, collagen type IV alpha 3 chain; COL4A4, collagen type IV alpha 4 chain; COL18A1, collagen type XVIII alpha 1 chain; CQ, chloroquine; GelMA, gelatin methacryloyl hydrogel; GO, Gene Ontology; GSEA, gene set enrichment analysis; HIF1A, hypoxia inducible factor 1 subunit alpha; HPLC, high-performance liquid chromatography; ITGAM/CD11B, integrin subunit alpha M; ITGAX/CD11C, integrin subunit alpha X; ITGB1/CdD9, integrin subunit beta 1; KEGG, Kyoto Encyclopedia of Genes and Genomes; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; micro-CT, microcomputed tomography; MSCs, mesenchymal stem cells; MTOR, mechanistic target of rapamycin kinase; NP, negative pressure; NPWT, negative pressure wound therapy; PRKAA1/AMPKα1, protein kinase AMP-activated catalytic subunit alpha 1; PRKAA2, protein kinase AMP-activated catalytic subunit alpha 2; PTPRC/CD45, protein tyrosine phosphatase receptor type C; ROS, reactive oxygen species; RUNX2, RUNX family transcription factor 2; SBI, SBI-0206965; SPP1/OPN, secreted phosphoprotein 1; THY1/CD90, Thy-1 cell surface antigen; SQSTM1, sequestosome 1; TGFB3, transforming growth factor beta 3; ULK1/Atg1, unc-51 like autophagy activating kinase 1.

Three-dimensional microenvironmental priming of human mesenchymal stem cells in hydrogels facilitates efficient and rapid retroviral gene transduction via accelerated cell cycle synchronization

There are numerous approaches to improve the low transduction efficiency of retroviral vectors in two-dimensional (2D) cell culture substrates. However, the effect of a three-dimensional (3D) microenvironment, which better mimics in vivo conditions, is unknown. Cytocompatible hyaluronic acid (HA) hydrogels are a good candidate to study this issue. Here, photocrosslinkable HA hydrogels with an elastic modulus of 1.0–2.7 kPa were successfully prepared by varying the degree of methacrylation in the HA backbone. Culturing human adipose-derived stem cells (hASCs) in a 3D microenvironment significantly reduces the amount of time required for retroviral gene transduction compared with the conventional 2D method and maintains a high transduction efficiency. This acceleration of retroviral gene transduction correlates with the rate of cell-cycle synchronization. hASCs cultured in a 3D microenvironment have a shorter G1 phase and total cell-cycle length than hASCs cultured using the conventional 2D method. This cell-cycle regulation is dependent on expression of cyclin D1. In summary, prior culturing of hASCs in a 3D microenvironment accelerates retroviral gene transduction by regulating cyclin D1 expression and accelerating cell-cycle synchronization. We conclude that priming via culturing in a 3D microenvironment facilitates efficient and rapid retroviral gene transduction of hASCs without inducing apoptosis.

New injectable two-step forming hydrogel for delivery of bioactive substances in tissue regeneration.

A hydrogel based on chitosan, collagen, hydroxypropyl-γ-cyclodextrin and polyethylene glycol was developed and characterized. The incorporation of nano-hydroxyapatite and pre-encapsulated hydrophobic/hydrophilic model drugs diminished the porosity of hydrogel from 81.62 ± 2.25% to 69.98 ± 3.07%. Interactions between components of hydrogel, demonstrated by FTIR spectroscopy and rheology, generated a network that was able to trap bioactive components and delay the burst delivery. The thixotropic behavior of hydrogel provided adaptability to facilitate its implantation in a minimally invasive way. Release profiles from microspheres included or not in hydrogel revealed a two-phase behavior with a burst- and a controlled-release period. The same release rate for microspheres included or not in the hydrogel in the controlled-release period demonstrated that mass transfer process was controlled by internal diffusion. Effective diffusion coefficients, Deff, that describe internal diffusion inside microspheres, and mass transfer coefficients, h, i.e. the contribution of hydrogel to mass transfer, were determined using ‘genetic algorithms’, obtaining values between 2.64·10−15 and 6.67·10−15 m2/s for Deff and 8.50·10−10 to 3.04·10−9 m/s for h. The proposed model fits experimental data, obtaining an R2-value ranged between 95.41 and 98.87%. In vitro culture of mesenchymal stem cells in hydrogel showed no manifestations of intolerance or toxicity, observing an intense proliferation of the cells after 7 days, being most of the scaffold surface occupied by living cells.

Multifunctional nanoparticles for intracellular drug delivery and photoacoustic imaging of mesenchymal stem cells.

Strategies that control the differentiation of mesenchymal stem cells (MSC) and enable image-guided cell implantation and longitudinal monitoring could advance MSC-based therapies for bone defects and injuries. Here we demonstrate a multifunctional nanoparticle system that delivers resveratrol (RESV) intracellularly to improve osteogenesis and enables photoacoustic imaging of MSCs. RESV-loaded nanoparticles (RESV-NPs), formulated from poly (lactic-co-glycolic) acid and iron oxide, enhanced the stability of RESV by 18-fold and served as photoacoustic tomography (PAT) contrast for MSCs. Pre-loading MSCs with RESV-NP upregulated RUNX2 expression with a resultant increase in mineralization by 27% and 45% compared to supplementation with RESV-NP and free RESV, respectively, in 2-dimensional cultures. When grown in polyethylene glycol-based hydrogels, MSCs pre-loaded with RESV-NPs increased the overall level and homogeneity of mineralization compared to those supplemented with free RESV or RESV-NP. The PAT detected RESV-NP-loaded MSCs with a resolution of 1500 cells/μL, which ensured imaging of MSCs upon encapsulation in a PEG-based hydrogel and implantation within the rodent cranium. Significantly, RESV-NP-loaded MSCs in hydrogels did not show PAT signal dilution over time or a reduction in signal upon osteogenic differentiation. This multifunctional NP platform has the potential to advance translation of stem cell-based therapies, by improving stem cell function and consistency via intracellular drug delivery, and enabling the use of a promising emerging technology to monitor cells in a clinically relevant manner.

Polyamidoamine dendrimer-PEG hydrogel and its mechanical property on differentiation of mesenchymal stem cells.

Biocompatible hydrogel systems with tunable mechanical properties have been reported to influence the behavior and differentiation of mesenchymal stem cells (MSCs). To develop a functionalized hydrogel system with well-defined chemical structures and tunable mechanical property for regulation of stem cell differentiation. An in situ-forming hydrogel system is developed by crosslinking vinyl sulfone functionalized polyamidoamine (PAMAM) dendrimer and multi-armed thiolated polyethylene glycol (PEG) through a thiol-ene Michael addition in aqueous conditions. The viability and differentiation of MSCs in hydrogels of different stiffness are conducted for 21 days under corresponding induction media. MSCs are viable in synthesized hydrogels after 48 hours of culture. By varying the concentrations of PAMAM dendrimer and PEG, hydrogels of different gelation time and stiffness are achieved. The MSC differentiation indicates that more osteogenic differentiation is observed in hard gel (5,663 Pa) and more adipogenic differentiation is observed in soft gel (77 Pa) in addition to the differentiation caused by each individual induction media during the process of culture. A biocompatible in situ-forming hydrogel system is successfully synthesized. This hydrogel system has shown influences on differentiation of MSCs and may potentially be important in developing therapeutic strategies in medical applications.

Culture of Mesenchymal Stem Cells in a Hydrogel Model of Vocal Fold Lamina Propria.

Stem cell injection has been proposed as an alternative approach for the restoration of vocal fold (VF) function in patients with VF scarring. To assess the therapeutic efficacy of this treatment strategy, we evaluated the behaviors of human mesenchymal stem cells (hMSCs) in hydrogels derived from thiolated hyaluronic acid (HA-SH) and poly(ethylene glycol) diacrylate (PEG-DA) entrapping assembled collagen fibrils (abbreviated as HPC gels). Three hydrogel formulations with varying amounts of collagen (0, 1 and 2 mg/mL) but a fixed HA-SH (5 mg/mL) and PEG-DA (2 mg/mL) concentration, designated as HPC0, HPC1 and HPC2, were investigated. The HPC gels exhibit similar pore sizes (35-50 nm) and AFM indentation moduli (~175 Pa), although the elastic shear modulus for HPC1 (~32 Pa) is lower than HPC0 and HPC2 (~55 Pa). Although HPC1 and HPC2 gels both promoted the development of an elongated cell morphology, greater cell spreading was observed in HPC2 than in HPC1 by day 7. At the transcript level, cells cultured in HPC1 and HPC2 gels had an increased expression of fibronectin and integrin β1, but a decreased expression of tissue inhibitor of metalloproteinase-1, collagen types I/III and HA synthase-1 when compared to cells cultured in HPC0 gels. Cellular expression of connective tissue growth factor was also elevated in HPC1 and HPC2 cultures. Importantly, the HPC2 hydrogels promoted a signficant up-regulation of matrix metalloproteinase 1, transforming growth factor β1, and epithelial growth factor receptor, indicating an increased tissue turnover. Overall, hMSCs cultured in HPC2 gels adopt a phenotype reminiscent of cells involved in the wound healing process, providing a platform to study the effectiveness of therapeutic stem cell treatments for VF scarring.

IIB or not IIB: development of a screening method by flow cytometry to evaluate correct chondrogenic conversion of human mesenchymal stem cells in hydrogel

Purpose: The use of mesenchymal stem cells (MSCs) represents a promising alternative cellular model to chondrocytes for articular cartilage repair but satisfactory protocols allowing proper chondrogenic conversion together with sufficient cartilage matrix production are still missing. The purpose of this study was to undertake an extensive characterization of human MSCs isolated from Wharton’s jelly (WJ), adipose tissue (AT), dental pulp (DP) and bone marrow (BM), and to confront their chondrogenic potential in serum-free culture conditions. Methods: MSCs were isolated and expanded in serum-free conditions. Their proliferation kinetics were evaluated. An extensive immunophenotyping analysis by flow cytometry was undertaken at the end of their first passage (P1). We then explored their potential to engage into adipogenic, osteogenic and chondrogenic lineages, the latter being assessed in serum-free conditions with a view of clinical application. This chondrogenic conversion was performed in micromass pellets and in another cellular model using MSCs embedded in agarose hydrogel. This model offers two advantages: 1) it mimicks a cartilage tissue engineering protocol where MSCs converted into chondrocytes and embedded in hydrogel form a cartilage gel that could be used to treat cartilage lesions and 2) the agarose nature of the hydrogel allows the release of the cells from the gel (by enzymatic digestion) at the end of their chondrogenic conversion, giving therefore the possibility to analyze them directly by flow cytometry. Recently, we developed and characterized the first antibody capable of detecting the IIB isoform of human type II procollagen, the only isoform of this collagen type that is expressed by well-differentiated chondrocytes. Here, we used this antibody by flow cytometry to look for intracellular IIB procollagen expression in MSCs undergoing chondrocyte differentiation in hydrogel. Results: The mean population doubling times between P1 and P5 ranged between 30 h and 55 h, depending on the tissue origin of MSCs. The doubling times remained constant from P1 to P5 and a minimal reservoir of 300 × 10ˆ6 cells (e.g., BM-MSCs) could be obtained after 5 passages, starting with 1 × 10ˆ6 cells harvested at the end of P1. Importantly, no karyotype abnormalities were detected after 5 passages. In brief, all cell sources yielded clinical-scale amounts of MSCs after expansion in serum-free culture conditions. By using an original panel of 27 surface markers that were analyzed by 8-color flow cytometry, our results revealed that all MSC sources were positive for the expression of the ISCT-validated markers (CD73, CD90, CD105) as well as for other recognized MSC markers (CD44). Nevertheless, differences in expression for other markers were found between the sources. BM-MSCs were the only cells to include subpopulations positive for all the putative skeletal markers analyzed (CD29, CD56, MSCA-1, Stro-1, CD106, CD146, CD271). All cell sources showed signs of adipogenic, osteogenic and chondrogenic conversion in monolayer cultures, after amplification in serum-free conditions. However, only BM-MSCs were able to clearly convert into true chondrocytes in micromass pellets or hydrogel cultures, as attested in particular by the expression of type IIB procollagen, monitored by immunohistochemistry or flow cytometry. Conclusions: It is possible to drive MSCs to the chondrogenic lineage in hydrogel after expansion and differentiation in serum-free conditions. BM-MSCs appear to be the best candidates for chondrogenic conversion in the conditions used in this study and this is in concordance with the largest panel of putative skeletal markers revealed by flow cytometry analysis for this category of MSCs. In this context, we have introduced an innovative quality control of the chondrocytic status of MSCs embedded in hydrogel, via measurement by flow cytometry of intracellular IIB procollagen, a marker of true chondrocytes.

Proliferation and osteogenic differentiation of mesenchymal stem cells in hydrogels of human blood plasma

The use of mesenchymal stem cells in clinical practice has increased considerably in the last decade because they play a supporting role in the processes of tissue repair and regeneration, becoming the main tool of cell therapy for the treatment of diseases functionally affecting bone and cartilage tissue . To evaluate in vitro the proliferative and osteogenic differentiation ability of mesenchymal stem cells derived from human adipose tissue in a blood plasma hydrogel. Mesenchymal stem cells were obtained from human adipose tissue explants and characterized by flow cytometry. Their multipotentiality was demonstrated by their ability to differentiate to adipogenic and osteogenic lineages. Cell proliferation and osteogenic differentiation ability of the cells cultured in blood plasma hydrogels were also evaluated. Mesenchymal stem cells derived from human adipose tissue growing in human blood plasma hydrogels showed a pattern of proliferation similar to that of the cells cultured in monolayer and also maintained their ability to differentiate to osteogenic lineage. Human blood plasma hydrogels are a suitable support for proliferation and osteogenic differentiation of mesenchymal stem cells derived from human adipose tissue and provides a substrate that is autologous, biocompatible, reabsorbable, easy to use, potentially injectable and economic, which could be used as a successful strategy for the management and clinical application of cell therapy in regenerative medicine.

Osteogenic differentiation of human bone marrow mesenchymal stem cells in hydrogel containing nacre powder

Nacre (or mother of pearl) can facilitate bone cell differentiation and can speed up their mineralization. Here we report on the capability of nacre to induce differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) and the production of extracellular matrix. hBM-MSCs were encapsulated in an alginate hydrogel containing different concentrations of powdered nacre and cultured in the same environment until Day 28. Analysis of osteogenic gene expression, histochemistry, second harmonic generation (SHG) microscopy, and Raman scattering spectroscopy were used to characterize the synthesis of the extracellular matrix. In the presence of nacre powder, a significant increase in matrix synthesis from D21 in comparison with pure alginate was observed. Histochemistry revealed the formation of a new tissue composed of collagen fibers in the presence of nacre (immunostaining and SHG), and hydroxyapatite crystals (Raman) in the alginate beads. These results suggest that nacre is efficient in hBM-MSCs differentiation, extracellular matrix production and mineralization in alginate 3D biomaterials.

Cartilage Matrix Formation by Bovine Mesenchymal Stem Cells in Three-dimensional Culture Is Age-dependent

Cartilage degeneration is common in the aged, and aged chondrocytes are inferior to juvenile chondrocytes in producing cartilage-specific extracellular matrix. Mesenchymal stem cells (MSCs) are an alternative cell type that can differentiate toward the chondrocyte phenotype. Aging may influence MSC chondrogenesis but remains less well studied, particularly in the bovine system. The objectives of this study were (1) to confirm age-related changes in bovine articular cartilage, establish how age affects chondrogenesis in cultured pellets for (2) chondrocytes and (3) MSCs, and (4) determine age-related changes in the biochemical and biomechanical development of clinically relevant MSC-seeded hydrogels. Native bovine articular cartilage from fetal (n = 3 donors), juvenile (n = 3 donors), and adult (n = 3 donors) animals was analyzed for mechanical and biochemical properties (n = 3-5 per donor). Chondrocyte and MSC pellets (n = 3 donors per age) were cultured for 6 weeks before analysis of biochemical content (n = 3 per donor). Bone marrow-derived MSCs of each age were also cultured within hyaluronic acid hydrogels for 3 weeks and analyzed for matrix deposition and mechanical properties (n = 4 per age). Articular cartilage mechanical properties and collagen content increased with age. We observed robust matrix accumulation in three-dimensional pellet culture by fetal chondrocytes with diminished collagen-forming capacity in adult chondrocytes. Chondrogenic induction of MSCs was greater in fetal and juvenile cell pellets. Likewise, fetal and juvenile MSCs in hydrogels imparted greater matrix and mechanical properties. Donor age and biochemical microenvironment were major determinants of both bovine chondrocyte and MSC functional capacity. In vitro model systems should be evaluated in the context of age-related changes and should be benchmarked against human MSC data.

In Vivo Chondrogenesis of Mesenchymal Stem Cells in a Photopolymerized Hydrogel

Surgical options for cartilage reconstruction can be significantly improved through advances in cartilage tissue engineering, whereby functional tissue replacements are created by growing cells on polymer scaffolds. The objective of this study was to use a photopolymerizable hydrogel to implant bone marrow-derived mesenchymal stem cells subcutaneously in a minimally invasive manner and promote cartilage tissue formation by the cells in vivo. Athymic nude mice were injected subcutaneously with polymer solutions of poly(ethylene) oxide diacrylate containing mesenchymal stem cells and placed under a UVA lamp to transdermally photopolymerize (solidify) the injected liquid. Experimental groups included polymer solutions with hyaluronic acid (HA), transforming growth factor (TGF)-beta3, or both. After 3 weeks of implantation, cartilage formation was evaluated by gene expression analysis and histologic techniques. Hyaluronic acid increased the viscosity of the polymer solutions, which helped maintain the injections at the desired site during photopolymerization. Mesenchymal stem cells in hydrogels containing both HA and TGF-beta3 produced the highest quality cartilage, based on expression of the cartilage-specific genes and production of proteoglycan and collagen II. When used independently, TGF-beta3 and HA alone induced cartilage-specific gene expression and collagen type II production; however, TGF-beta3 was essential for proteoglycan production. HA enhanced proteoglycan production when combined with TGF-beta3 and reduced expression and production of collagen I. This study is the first to demonstrate the minimally invasive implantation and subsequent chondrogenic differentiation of mesenchymal stem cells in the subcutaneous environment. This lays the foundation for further optimization of a novel and practical technology for cartilage reconstruction.