Human mesenchymal stem cell-derived matrices for enhanced osteoregeneration
ABSTRACT: The methodology for the repair of critical-sized or non-union bone lesions has unpredictable efficacy due in part to our incomplete knowledge of bone repair and the biocompatibility of bone substitutes. Although human mesenchymal stem cells (hMSCs) differentiate into osteoblasts, which promote bone growth, their ability to repair bone has been unpredictable. We hypothesized that given the multi-stage process of osteogenesis, hMSC-mediated repair might be maximal at a specific time-point of healing. Utilizing a mouse model of calvarial healing, we demonstrate that the osteo-repair capacity of hMSCs can be substantially augmented by treatment with an inhibitor of peroxisome-proliferator-activated-receptor-γ, but efficacy is confined to the rapid osteogenic phase. Upon entry into the bone-remodeling phase, hMSC retention signals are lost, resulting in truncation of healing. To solve this limitation, we prepared a scaffold consisting of hMSC-derived extracellular matrix (ECM) containing the necessary biomolecules for extended site-specific hMSC retention. When inhibitor-treated hMSCs were co-administered with ECM, they remained at the injury well into the remodeling phase of healing, which resulted in reproducible and complete repair of critical-sized defects in 3 weeks. These data suggest that hMSC-derived ECM and inhibitor-treated hMSCs could be employed at optimal times to substantially and reproducibly improve bone repair. To gain insight into the superior healing potential of GW-hMSCs and also what might be accounting for their extended engraftment, microarray analyses on the RNA extracted from the calvarial tissue recovered after days 5 and 14 were performed. Equal amounts of total RNA from 4 animals per group and time point were pooled and animals receiving control (DMSO) or peroxisome proliferator-activated receptor-gamma inhibitor GW9662-treated hMSCs were compared with the assumption that murine cross-hybridization would be constant throughout the samples and thus be subtracted from the analysis.
Project description:Human mesenchymal stem cells (hMSCs) from bone marrow are regarded as putative osteoblast progenitors in vivo and differentiate into osteoblasts in vitro. Positive signaling by the canonical wingless (Wnt) pathway is critical for the differentiation of MSCs into osteoblasts. In contrast, activation of the peroxisome proliferator-activated receptor-gamma (PPARgamma)-mediated pathway results in adipogenesis. We therefore compared the effect of glycogen-synthetase-kinase-3beta (GSK3beta) inhibitors and PPARgamma inhibitors on osteogenesis by hMSCs. Both compounds altered the intracellular distribution of beta-catenin and GSK3beta in a manner consistent with activation of Wnt signaling. With osteogenic supplements, the GSK3beta inhibitor 6-bromo-indirubin-3'-oxime (BIO) and the PPARgamma inhibitor GW9662 (GW) enhanced early osteogenic markers, alkaline phosphatase (ALP), and osteoprotegerin (OPG) by hMSCs and transcriptome analysis demonstrated up-regulation of genes encoding bone-related structural proteins. At higher doses of the inhibitors, ALP levels were attenuated, but dexamethasone-induced biomineralization was accelerated. When hMSCs were pretreated with BIO or GW and implanted into experimentally induced nonself healing calvarial defects, GW treatment substantially increased the capacity of the cells to repair the bone lesion, whereas BIO treatment had no significant effect. Further investigation indicated that unlike GW, BIO induced cell cycle inhibition in vitro. Furthermore, we found that GW treatment significantly reduced expression of chemokines that may exacerbate neutrophil- and macrophage-mediated cell rejection. These data suggest that use of PPARgamma inhibitors during the preparation of hMSCs may enhance the capacity of the cells for osteogenic cytotherapy, whereas adenine analogs such as BIO can adversely affect the viability of hMSC preparations in vitro and in vivo.
Project description:RNA interference (RNAi) may be an effective and valuable tool for promoting the growth of functional tissue, as short interfering RNA (siRNA) and microRNA (miRNA) can block the expression of genes that have negative effects on tissue regeneration. Our group has recently reported that the localized and sustained presentation of siRNA against noggin (siNoggin) and miRNA-20a from in situ forming poly(ethylene glycol) (PEG) hydrogels enhanced osteogenic differentiation of encapsulated human bone marrow-derived mesenchymal stem cells (hMSCs). Here, the capacity of the hydrogel system to accelerate bone formation in a rat calvarial bone defect model is presented. After 12?weeks post-implantation, the hydrogels containing encapsulated hMSCs and miRNA-20a resulted in more bone formation in the defects than the hydrogels containing hMSCs without siRNA or with negative control siRNA. This localized and sustained RNA interfering molecule delivery system may provide an excellent platform for healing bony defects and other tissues. STATEMENT OF SIGNIFICANCE:Delivery of RNAi molecules may be a valuable strategy to guide cell behavior for tissue engineering applications, but to date there have been no reports of a biomaterial system capable of both encapsulation of cells and controlled delivery of incorporated RNA. Here, we present PEG hydrogels that form in situ via Michael type reaction, and that permit encapsulation of hMSCs and the concomitant controlled delivery of siNoggin and/or miRNA-20a. These RNAs were chosen to suppress noggin, a BMP-2 antagonist, and/or PPAR-?, a negative regulator of BMP-2-mediated osteogenesis, and therefore promote osteogenic differentiation of hMSCs and subsequent bone repair in critical-sized rat calvarial defects. Simultaneous delivery of hMSCs and miRNA-20a enhanced repair of these defects compared to hydrogels containing hMSCs without siRNA or with negative control siRNA. This in situ forming PEG hydrogel system offers an exciting platform for healing critical-sized bone defects by localized, controlled delivery of RNAi molecules to encapsulated hMSCs and surrounding cells.
Project description:Stem cell therapies are limited by poor cell survival and engraftment. A hurdle to the use of materials for cell delivery is the lack of understanding of material properties that govern transplanted stem cell functionality. Here, we show that synthetic hydrogels presenting integrin-specific peptides enhance the survival, persistence, and osteo-reparative functions of human bone marrow-derived mesenchymal stem cells (hMSCs) transplanted in murine bone defects. Integrin-specific hydrogels regulate hMSC adhesion, paracrine signaling, and osteoblastic differentiation in vitro. Hydrogels presenting GFOGER, a peptide targeting ?2?1 integrin, prolong hMSC survival and engraftment in a segmental bone defect and result in improved bone repair compared to other peptides. Integrin-specific hydrogels have diverse pleiotropic effects on hMSC reparative activities, modulating in vitro cytokine secretion and in vivo gene expression for effectors associated with inflammation, vascularization, and bone formation. These results demonstrate that integrin-specific hydrogels improve tissue healing by directing hMSC survival, engraftment, and reparative activities.
Project description:Most bones of the human body form and heal through endochondral ossification, whereby hypertrophic cartilage (HyC) is formed and subsequently remodeled into bone. We previously demonstrated that HyC can be engineered from human mesenchymal stromal cells (hMSC), and subsequently devitalized by apoptosis induction. The resulting extracellular matrix (ECM) tissue retained osteoinductive properties, leading to ectopic bone formation. In this study, we aimed at engineering and devitalizing upscaled quantities of HyC ECM within a perfusion bioreactor, followed by in vivo assessment in an orthotopic bone repair model. We hypothesized that the devitalized HyC ECM would outperform a clinical product currently used for bone reconstructive surgery. Human MSC were genetically engineered with a gene cassette enabling apoptosis induction upon addition of an adjuvant. Engineered hMSC were seeded, differentiated, and devitalized within a perfusion bioreactor. The resulting HyC ECM was subsequently implanted in a 10-mm rabbit calvarial defect model, with processed human bone (Maxgraft®) as control. Human MSC cultured in the perfusion bioreactor generated a homogenous HyC ECM and were efficiently induced towards apoptosis. Following six weeks of in vivo implantation, microcomputed tomography and histological analyses of the defects revealed an increased bone formation in the defects filled with HyC ECM as compared to Maxgraft®. This work demonstrates the suitability of engineered devitalized HyC ECM as a bone substitute material, with a performance superior to a state-of-the-art commercial graft. Streamlined generation of the devitalized tissue transplant within a perfusion bioreactor is relevant towards standardized and automated manufacturing of a clinical product.
Project description:Microenvironment extracellular matrices (ECMs) influence cell adhesion, proliferation and differentiation. The ECMs of different microenvironments have distinctive compositions and architectures. This investigation addresses effects ECMs deposited by a variety of cell types and decellularized with a cold-EDTA protocol have on multipotent human mesenchymal stromal/stem cell (hMSC) behavior and differentiation. The cold-EDTA protocol removes intact cells from ECM, with minimal ECM damage and contamination. The decellularized ECMs deposited by cultured hMSCs, osteogenic hMSCs, and two smooth muscle cell (SMC) lines were tested for distinctive effects on the behavior and differentiation of early passage ('naïve') hMSC plated and cultured on the decellularized ECMs. Uninduced hMSC decellularized ECM enhanced naïve hMSC proliferation and cell motility while maintaining stemness. Decellularized ECM deposited by osteogenic hMSCs early in the differentiation process stimulated naïve hMSCs osteogenesis and substrate biomineralization in the absence of added dexamethasone, but this osteogenic induction potential was lower in ECMs decellularized later in the osteogenic hMSC differentiation process. Decellularized ECMs deposited by two smooth muscle cell lines induced naïve hMSCs to become smooth muscle cell-like with distinctive phenotypic characteristics of contractile and synthetic smooth muscle cells. This investigation demonstrates a useful approach for obtaining functional cell-deposited ECM and highlights the importance of ECM specificity in influencing stem cell behavior.
Project description:Hydrophilic poly(ethylene glycol) diacrylate (PEGDA) hydrogel surfaces resist protein adsorption and are generally thought to be unsuitable for anchorage-dependent cells to adhere. Intriguingly, our previous findings revealed that PEGDA superporous hydrogel scaffolds (SPHs) allow anchorage of bone marrow derived human mesenchymal stem cells (hMSCs) and support their long-term survival. Therefore, we hypothesized that the physicochemical characteristics of the scaffold impart properties that could foster cellular responses. We examined if hMSCs alter their microenvironment to allow cell attachment by synthesizing their own extracellular matrix (ECM) proteins. Immunofluorescence staining revealed extensive expression of collagen type I, collagen type IV, laminin, and fibronectin within hMSC-seeded SPHs by the end of the third week. Whether cultured in serum-free or serum-supplemented medium, hMSC ECM protein gene expression patterns exhibited no substantial changes. The presence of serum proteins is required for initial anchorage of hMSCs within the SPHs but not for the hMSC survival after 24 h. In contrast to 2D expansion on tissue culture plastic (TCP), hMSCs cultured within SPHs proliferate similarly in the presence or absence of serum. To test whether hMSCs retain their undifferentiated state within the SPHs, cell-seeded constructs were cultured for 3 weeks in stem cell maintenance medium and the expression of hMSC-specific cell surface markers were evaluated by flow cytometry. CD105, CD90, CD73, and CD44 were present to a similar extent in the SPH and in 2D monolayer culture. We further demonstrated multilineage potential of hMSCs grown in the PEGDA SPHs, whereby differentiation into osteoblasts, chondrocytes, and adipocytes could be induced. The present study demonstrates the potential of hMSCs to alter the "blank" PEGDA environment to a milieu conducive to cell growth and multilineage differentiation by secreting adhesive ECM proteins within the porous network of the SPH scaffolds.
Project description:Background:Human mesenchymal stem cells (hMSCs) are, due to their pluripotency, useful sources of cells for stem cell therapy and tissue regeneration. The phenotypes of hMSCs are strongly influenced by their microenvironment, in particular the extracellular matrix (ECM), the composition and structure of which are important in regulating stem cell fate. In reciprocal manner, the properties of ECM are remodeled by the hMSCs, but the mechanism involved in ECM remodeling by hMSCs under topographical stimulus is unclear. In this study, we therefore examined the effect of nanotopography on the expression of ECM proteins by hMSCs by analyzing the quantity and structure of the ECM on a nanogrooved surface. Methods:To develop the nanoengineered, hMSC-derived ECM, we fabricated the nanogrooves on a coverglass using a UV-curable polyurethane acrylate (PUA). Then, hMSCs were cultivated on the nanogrooves, and the cells at the full confluency were decellularized. To analyze the effect of nanotopography on the hMSCs, the hMSCs were re-seeded on the nanoengineered, hMSC-derived ECM. Results:hMSCs cultured within the nano-engineered hMSC-derived ECM sheet showed a different pattern of expression of ECM proteins from those cultured on ECM-free, nanogrooved surface. Moreover, hMSCs on the nano-engineered ECM sheet had a shorter vinculin length and were less well-aligned than those on the other surface. In addition, the expression pattern of ECM-related genes by hMSCs on the nanoengineered ECM sheet was altered. Interestingly, the expression of genes for osteogenesis-related ECM proteins was downregulated, while that of genes for chondrogenesis-related ECM proteins was upregulated, on the nanoengineered ECM sheet. Conclusions:The nanoengineered ECM influenced the phenotypic features of hMSCs, and that hMSCs can remodel their ECM microenvironment in the presence of a nanostructured ECM to guide differentiation into a specific lineage.
Project description:Shockwave treatment promotes bone healing of nonunion fractures. In this study, we investigated whether this effect could be due to adenosine 5'-triphosphate (ATP) release-induced differentiation of human mesenchymal stem cells (hMSCs) into osteoprogenitor cells. Cultured bone marrow-derived hMSCs were subjected to shockwave treatment and ATP release was assessed. Osteogenic differentiation and mineralization of hMSCs were evaluated by examining alkaline phosphatase activity, osteocalcin production, and calcium nodule formation. Expression of P2X7 receptors and c-fos and c-jun mRNA was determined with real-time reverse transcription polymerase chain reaction and Western blotting. P2X7-siRNA, apyrase, P2 receptor antagonists, and p38 MAPK inhibitors were used to evaluate the roles of ATP release, P2X7 receptors, and p38 MAPK signaling in shockwave-induced osteogenic hMSCs differentiation. Shockwave treatment released significant amounts (? 7 ?M) of ATP from hMSCs. Shockwaves and exogenous ATP induced c-fos and c-jun mRNA transcription, p38 MAPK activation, and hMSC differentiation. Removal of ATP with apyrase, targeting of P2X7 receptors with P2X7-siRNA or selective antagonists, or blockade of p38 MAPK with SB203580 prevented osteogenic differentiation of hMSCs. Our findings indicate that shockwaves release cellular ATP that activates P2X7 receptors and downstream signaling events that caused osteogenic differentiation of hMSCs. We conclude that shockwave therapy promotes bone healing through P2X7 receptor signaling, which contributes to hMSC differentiation.
Project description:Integrins provide the primary link between mesenchymal stem cells (MSCs) and their surrounding extracellular matrix (ECM), with different integrin pairs having specificity for different ECM molecules or peptide sequences contained within them. It is widely acknowledged that the type of ECM present can influence MSC differentiation; however, it is yet to be determined how specific integrin-ECM interactions may alter this or how they change during differentiation. We determined that human bone marrow-derived mesenchymal stem cells (hMSCs) express a broad range of integrins in their undifferentiated state and show a dramatic, but transient, increase in the level of ?5 integrin on day 7 of osteogenesis and an increase in ?6 integrin expression throughout adipogenesis. We used a nonfouling polystyrene-block-poly(ethylene oxide)-copolymer (PS-PEO) surface to present short peptides with defined integrin-binding capabilities (RGD, IKVAV, YIGSR, and RETTAWA) to hMSCs and investigate the effects of such specific integrin-ECM contacts on differentiation. hMSCs cultured on these peptides displayed different morphologies and had varying abilities to differentiate along the osteogenic and adipogenic lineages. The peptide sequences most conducive to differentiation (IKVAV for osteogenesis and RETTAWA and IKVAV for adipogenesis) were not necessarily those that were bound by those integrin subunits seen to increase during differentiation. Additionally, we also determined that presentation of RGD, which is bound by multiple integrins, was required to support long-term viability of hMSCs. Overall we confirm that integrin-ECM contacts change throughout hMSC differentiation and show that surfaces presenting defined peptide sequences can be used to target specific integrins and ultimately influence hMSC differentiation. This platform also provides information for the development of biomaterials capable of directing hMSC differentiation for use in tissue engineering therapies.
Project description:Self-sustainability after implantation is one of the critical obstacles facing large engineered tissues. A preformed functional vascular network provides an effective solution for solving the mass transportation problem. With the support of mural cells, endothelial cells (ECs) can form microvessels within engineered tissues. As an important mural cell, human mesenchymal stem cells (hMSCs) not only stabilize the engineered microvessel network, but also preserve their multi-potency when grown under optimal culture conditions. A prevascularized hMSC/extracellular matrix (ECM) sheet fabricated by the combination of hMSCs, ECs and a naturally derived nanofibrous ECM scaffold offers great opportunity for engineering mechanically strong and completely biological three-dimensional prevascularized tissues. The objective of this study was to create a prevascularized hMSC/ECM sheet by co-culturing ECs and hMSCs on a nanofibrous ECM scaffold. Physiologically low oxygen (2% O2 ) was introduced during the 7 day hMSC culture to preserve the stemness of hMSCs and thereby their capability to secrete angiogenic factors. The ECs were then included to form microvessels under normal oxygen (20% O2 ) for up to 7 days. The results showed that a branched and mature vascular network was formed in the co-culture condition. Angiogenic factors vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and angiopoietin-1 (Ang-1) were significantly increased by low-oxygen culture of hMSCs, which further stabilized and supported the maturation of microvessels. A differentiation assay of the prevascularized ECM scaffold demonstrated a retained hMSC multi-potency in the hypoxia cultured samples. The prevascularized hMSC/ECM sheet holds great promise for engineering three-dimensional prevascularized tissues for diverse applications.