The effect of collagen I mimetic peptides on mesenchymal stem cell adhesion and differentiation, and on bone formation at hydroxyapatite surfaces.

Integrin-binding peptides increase cell adhesion to naive hydroxyapatite (HA), however, in the body, HA becomes rapidly modified by protein adsorption. Previously we reported that, when combined with an adsorbed protein layer, RGD peptides interfered with cell adhesion to HA. In the current study we evaluated mesenchymal stem cell (MSC) interactions with HA disks coated with the collagen-mimetic peptides, DGEA, P15 and GFOGER. MSCs adhered equally well to disks coated with DGEA, P15, or collagen I, and all three substrates, but not GFOGER, supported greater cell adhesion than uncoated HA. When peptide-coated disks were overcoated with proteins from serum or the tibial microenvironment, collagen mimetics did not inhibit MSC adhesion, as was observed with RGD, however neither did they enhance adhesion. Given that activation of collagen-selective integrins stimulates osteoblastic differentiation, we monitored osteocalcin secretion and alkaline phosphatase activity from MSCs adherent to DGEA or P15-coated disks. Both of these osteoblastic markers were upregulated by DGEA and P15, in the presence and absence of differentiation-inducing media. Finally, bone formation on HA tibial implants was increased by the collagen mimetics. Collectively these results suggest that collagen-mimetic peptides improve osseointegration of HA, most probably by stimulating osteoblastic differentiation, rather than adhesion, of MSCs.

Heparan sulfate mediates the proliferation and differentiation of rat mesenchymal stem cells.

The growth and differentiation of mesenchymal stem cells (MSCs) is controlled by various growth factors, the activities of which can be modulated by heparan sulfates (HSs). We have previously noted the necessity of sulfated glycosaminoglycans for the fibroblast growth factor type 2 (FGF-2)-stimulated differentiation of osteoprogenitor cells. Here we show that exogenous application of HS to cultures of primary rat MSCs stimulates their proliferation, leading to increased expression of osteogenic markers and enhanced bone nodule formation. FGF-2 can also increase the proliferation, and osteogenic differentiation of rat bone marrow stem cells (rMSCs) when applied exogenously during their linear growth. However, as opposed to exogenous HS, the continuous use of FGF-2 during in vitro differentiation completely blocked rMSC mineralization. We show that the effects of both FGF-2 and HS are mediated through FGF receptor 1 (FGFR1) and that inhibition of signaling through this receptor arrests cell growth, resulting in the cells being unable to reach the critical density necessary to induce differentiation. Blocking FGFR1 signaling in postconfluent osteogenic cultures significantly increased calcium deposition. Taken together our data suggest that FGFR1 signaling plays an important role during osteogenic differentiation, first by stimulating cell growth that is closely followed by an inhibitory effect once the cells have reached confluence. It also confirms the importance of HS as a coreceptor for the signaling of endogenous FGF-2 and suggests that purified glycosaminoglycans may be attractive alternatives to growth factors for improved ex vivo growth and differentiation of MSCs.

Comparison of mesenchymal stem cell and osteosarcoma cell adhesion to hydroxyapatite.

Immortalized cells are often used to model the behavior of osteogenic cells on orthopaedic and dental biomaterials. In the current study we compared the adhesive behavior of two osteosarcoma cell lines, MG-63 and Saos-2, with that of mesenchymal stem cells (MSCs) on hydroxyapatite (HA). It was found that osteosarcoma cells demonstrated maximal binding to fibronectin-coated HA, while MSCs alternately preferred HA coated with collagen-I. Interesting, the binding of MG-63 and Saos-2 cells to fibronectin was mediated by both alpha5 and alphav-containing integrin heterodimers, whereas only alphav integrins were used by MSCs. Cell spreading was also markedly different for the three cell types. Osteosarcoma cells exhibited optimal spreading on fibronectin, but poor spreading on HA disks coated with fetal bovine serum. In contrast, MSCs spread very well on serum-coated surfaces, but less extensively on fibronectin. Finally, we evaluated integrin expression and found that MSCs have higher levels of alpha2 integrin subunits relative to MG-63 or Saos-2 cells, which may explain the enhanced adhesion of MSCs on collagen-coated HA. Collectively our results suggest that osteosarcoma cells utilize different mechanisms than MSCs during initial attachment to protein-coated HA, thereby calling into question the suitability of these cell lines as in vitro models for cell/biomaterial interactions.

The effect of RGD peptides on osseointegration of hydroxyapatite biomaterials.

Given that hydroxyapatite (HA) biomaterials are highly efficient at adsorbing proadhesive proteins, we questioned whether functionalizing HA with RGD peptides would have any benefit. In this study, we implanted uncoated or RGD-coated HA disks into rat tibiae for 30 min to allow endogenous protein adsorption, and then evaluated mesenchymal stem cell (MSC) interactions with the retrieved disks. These experiments revealed that RGD, when presented in combination with adsorbed tibial proteins (including fibronectin, vitronectin and fibrinogen), has a markedly detrimental effect on MSC adhesion and survival. Moreover, analyses of HA disks implanted for 5 days showed that RGD significantly inhibits total bone formation as well as the amount of new bone directly contacting the implant perimeter. Thus, RGD, which is widely believed to promote cell/biomaterial interactions, has a negative effect on HA implant performance. Collectively these results suggest that, for biomaterials that are highly interactive with the tissue microenvironment, the ultimate effects of RGD will depend upon how signaling from this peptide integrates with endogenous processes such as protein adsorption.

Autocrine fibroblast growth factor 2 increases the multipotentiality of human adipose-derived mesenchymal stem cells.

Multipotent mesenchymal stem cells (MSCs), first identified in the bone marrow, have subsequently been found in many other tissues, including fat, cartilage, muscle, and bone. Adipose tissue has been identified as an alternative to bone marrow as a source for the isolation of MSCs, as it is neither limited in volume nor as invasive in the harvesting. This study compares the multipotentiality of bone marrow-derived mesenchymal stem cells (BMSCs) with that of adipose-derived mesenchymal stem cells (AMSCs) from 12 age- and sex-matched donors. Phenotypically, the cells are very similar, with only three surface markers, CD106, CD146, and HLA-ABC, differentially expressed in the BMSCs. Although colony-forming units-fibroblastic numbers in BMSCs were higher than in AMSCs, the expression of multiple stem cell-related genes, like that of fibroblast growth factor 2 (FGF2), the Wnt pathway effectors FRAT1 and frizzled 1, and other self-renewal markers, was greater in AMSCs. Furthermore, AMSCs displayed enhanced osteogenic and adipogenic potential, whereas BMSCs formed chondrocytes more readily than AMSCs. However, by removing the effects of proliferation from the experiment, AMSCs no longer out-performed BMSCs in their ability to undergo osteogenic and adipogenic differentiation. Inhibition of the FGF2/fibroblast growth factor receptor 1 signaling pathway demonstrated that FGF2 is required for the proliferation of both AMSCs and BMSCs, yet blocking FGF2 signaling had no direct effect on osteogenic differentiation. Disclosure of potential conflicts of interest is found at the end of this article.

The effect of adsorbed serum proteins, RGD and proteoglycan-binding peptides on the adhesion of mesenchymal stem cells to hydroxyapatite.

Prior studies from our laboratory have shown that RGD peptides increase the attachment of mesenchymal stem cells (MSCs) to hydroxyapatite (HA), however, RGD does not induce cell spreading when coupled to this type of biomaterial. In an effort to improve MSC spreading, and possibly cell attachment, proteoglycan-binding peptides (KRSR or FHRRIKA) were combined with RGD in the current study. It was found that the peptide combinations did not enhance MSC attachment relative to RGD alone, although a slight amount of spreading was elicited by both KRSR and FHRRIKA. Similar results were obtained with proteoglycan-binding peptides modified with a heptaglutamate domain, a motif that improves peptide tethering to HA. To determine whether differentiation status affected cell responses, MSCs were in vitro differentiated into osteoblasts, and evaluated as before. These experiments revealed that, like MSCs, osteoblasts did not adhere in greater numbers to the peptide combinations. Finally, none of the peptides or peptide combinations were able to stimulate the robust amount of cell adhesion and spreading elicited by serum-coated HA surfaces (of note, five different species of serum were tested). Given the propensity of HA to adsorb proadhesive proteins from blood/serum, we question the utility of functionalizing HA with RGD and/or proteoglycan-binding peptides.

Primary human marrow stromal cells and Saos-2 osteosarcoma cells use different mechanisms to adhere to hydroxylapatite.

One important step in bone formation on hard tissue implants is adhesion of osteoblast precursors to the implant surface. In this study, we used function-blocking antibodies against integrin subunits to characterize the mechanisms used by human marrow stromal cells and Saos-2 osteosarcoma cells to adhere to protein-coated hydroxylapatite (HA). We found that Saos-2 use both alpha5- and alphav-containing integrins, whereas stromal cells use alphav-containing integrins but not alpha5-containing integrins, despite the presence of alpha5-containing integrins on cell surfaces. On the basis of this difference, we examined binding of these cell types to HA coated with fibronectin (FN) or vitronectin (VN), to determine whether these ligands for alpha5 and alphav integrins could enhance the numbers or morphology of cells adhered to them. We also examined the adhesion of cells to HA coated with RGD peptides designed to bind to FN or VN receptors. Morphology and number of adherent stromal cells were markedly enhanced on serum-coated surfaces compared with FN or VN alone, whereas, surprisingly, Saos-2 cells failed to spread on serum-coated HA and displayed superior spreading and stress fiber formation on FN-coated [corrected] HA. Collectively, these results have important implications for the design of protein coatings to enhance the performance of HA implants.

Mesenchymal stem cell adhesion and spreading on nanostructured biomaterials.

Bone marrow-derived human mesenchymal stem cells were seeded in serum-free media onto ion beam-deposited nanostructured metalloceramic (Ti-Cr-N) films and plasma-nitrided titanium disks, which were left uncoated as well as precoated with fetal bovine serum. Precoating the disks with serum appears to stimulate cell spreading on both the titanium nitride and metalloceramic materials for as little as 1 hour incubation time. The implication is that both of these materials can adsorb serum proteins in amounts sufficient to influence cell adhesion and spreading for potentially improved in vivo response of orthopedic and dental implants. The materials in this study may prove to exhibit enhanced biological and mechanical properties when compared to conventional micron-scale implant materials such as titanium or cobalt-chrome alloys.