Modulation of differentiation and mineralization of marrow stromal cells cultured on biomimetic hydrogels modified with Arg-Gly-Asp containing peptides

We synthesized biomimetic hydrogels modified with an osteopontin-derived peptide (ODP) and used them as a substrate for in vitro culture of marrow stromal cells (MSCs) to investigate the effect of the biomimetic surface on differentiation of MSCs into osteoblasts. Proliferation and biological assays for 16 days proved that MSCs became differentiated into osteoblasts secreting osteogenic phenotypic markers such as alkaline phosphatase (ALP), osteopontin, and mineralized calcium. In addition, there was an additive effect of the cell-binding peptide on differentiation and mineralization of MSCs cultured in the presence of soluble osteogenic supplements in cell culture media. For example, calcium content at day 16 on peptide-modified hydrogels was significantly higher than on tissue culture polystyrene. Two general trends were observed: (1) proliferation of MSCs decreased as the amount of differentiation markers increased, and (2) higher peptide concentrations accelerated the differentiation of MSCs. On the hydrogel modified with ODP, ALP activity exhibited a maximum value of 36.7 +/- 4.2 pmol/cell/h at day 10 for the concentration of 2 micromol/g while the culture time needed for maximum ALP activity occurred on day 13 for the lower concentrations. On the same hydrogel, the calcium content at day 10 was 21.4 +/- 2.3 ng/cell for the peptide concentration of 2 micromol/g and 1.0 +/- 0.3 ng/cell for 1.0 micromol/g. We used Gly-Arg-Gly-Asp-Ser (GRGDS) for modification of the hydrogel as a comparison to the results with ODP. However, osteoblast development was not significantly affected by the nature of the binding peptide sequences. These results suggest that MSC function can be modulated by variation of the peptide concentration in biomimetic hydrogels used for scaffold-based bone tissue engineering.     

Osteogenic differentiation of rat bone marrow stromal cells cultured on Arg-Gly-Asp modified hydrogels without dexamethasone and beta-glycerol phosphate

In this study, we investigated the effect of signaling peptides incorporated into oligo(poly(ethylene glycol) fumarate) (OPF) hydrogels on in vitro differentiation and mineralization of marrow stromal cells (MSCs) cultured in media without soluble osteogenic supplements (dexamethasone and beta-glycerol phosphate). When MSCs were cultured for 16 days on OPF hydrogels modified with Arg-Gly-Asp (RGD) containing peptides, the normalized cell number was dependent on the peptide concentration between days 0 and 5 and reached comparable values at day 10 regardless of the concentration. The alkaline phosphatase (ALP) activity of MSCs on the peptide-modified OPF hydrogels was also concentration-dependent: ALP activity showed peaks on day 10 or day 13 on OPF hydrogels modified with 2.0 and 1.0 micromol peptide/g, which were significantly greater than those on the OPF hydrogels modified with 0.1 micromol peptides/g or no peptide. A characteristic marker of osteoblastic differentiation, osteopontin (OPN), was detected for all the test groups. However, OPN secretion between days 0 and 10 was significantly higher on the peptide modified hydrogels compared to that on tissue culture-treated polystyrene. Taken together, the results indicate that the presence of signaling peptide allows for a favorable microenvironment for MSCs to differentiate into osteoblasts and produce mineralized matrix, although the soluble factors may further enhance calcium deposition. These findings further support the usefulness of OPF hydrogels as scaffolds for guided bone regeneration, and represent an initial step in exploring the complex relationship between soluble and insoluble factors in osteogenic differentiation on biodegradable materials.     

Effect of hydrogel porosity on marrow stromal cell phenotypic expression

This study describes investigation of porous photocrosslinked oligo[(polyethylene glycol) fumarate] (OPF) hydrogels as potential matrix for osteoblastic differentiation of marrow stromal cells (MSCs). The porosity and interconnectivity of porous hydrogels were assessed using magnetic resonance microscopy (MRM) as a noninvasive investigative tool that could image the water construct inside the hydrogels at a high-spatial resolution. MSCs were cultured onto the porous hydrogels and cell number was assessed using PicoGreen DNA assay. Our results showed 10% of cells initially attached to the surface of scaffolds. However, cells did not show significant proliferation over a time period of 14 days. MSCs cultured on porous hydrogels had increased alkaline phosphatase activity as well as deposition of calcium, suggesting successful differentiation and maturation to the osteoblastic phenotype. Moreover, continued expression of type I collagen and osteonectin over 14 days confirmed osteoblastic differentiation of MSCs. MRM was also applied to monitor osteogenesis of MSCs on porous hydrogels. MRM images showed porous scaffolds became consolidated with osteogenic progression of cell differentiation. These findings indicate that porous OPF scaffolds enhanced MSC differentiation leading to development of bone-like mineralized tissue.     

Spatial distribution of mineralized bone matrix produced by marrow mesenchymal stem cells in self-assembling peptide hydrogel scaffold

We evaluated the osteogenic differentiation of mesenchymal stem cells (MSCs) using a new class of synthetic self-assembling peptide hydrogels, RADA 16, as a scaffold for three-dimensional culture. MSCs derived from rat bone marrow were culture-expanded and seeded into the hydrogel and further cultured in osteogenic medium containing beta-glycerophosphate, ascorbic acid, and dexamethasone for 2-4 weeks. High alkaline phosphatase activity and osteocalcin (OC) contents were detected at both the protein and gene expression levels during the culture periods. Both calcium and the OC contents increased over time, indicating the growth of a mineralized extracellular matrix within the hydrogel. Moreover, the process of the growth of the mineralized matrix determined by three-dimensional microarchitecture images was obtained by confocal laser scanning microscopy. The findings show that MSCs can differentiate into mature osteoblasts to form mineralized matrices within the hydrogel scaffold. Importantly, the differentiation can occur three dimensionally within the hydrogel, indicating that RADA 16 can be considered attractive synthetic biomaterial for use in bone tissue engineering.     

Collagen three-dimensional hydrogel matrix carrying basic fibroblast growth factor for the cultivation of mesenchymal stem cells and osteogenic differentiation

Three-dimensional (3D) collagen hydrogels have been extensively used for cell culture experiments and are more closely representative of in vivo conditions than monolayer (2D) culture. Here we cultured rat bone marrow-derived mesenchymal stem cells (MSCs) in collagen hydrogels containing varying concentrations of basic fibroblast growth factor (bFGF) to examine the effect of bFGF on MSC proliferation and osteogenic differentiation in 3D culture. The optimal bFGF concentration that promoted the greatest degree of cell proliferation and expression of the early osteogenic induction marker alkaline phosphatase was also determined. Subsequent quantitative real-time polymerase chain reaction analysis of gene expression demonstrated that bFGF promoted significant upregulation of the bone-related genes: collagen type I, osteopontin (OPN), bone sialoprotein (BSP), and osteocalcin (OCN) for periods of up to 21 days. Immunofluorescence staining and fluorescence-activated cell sorting analysis further supported the enhanced osteogenic differentiation of cells as a greater proportion of cells were found to express OPN. Matrix mineralization within the collagen hydrogels was enhanced in the presence of bFGF, as assessed by calcium detection using von Kossa staining. These results clearly demonstrate a positive effect of bFGF on proliferation and osteogenic induction of MSCs in 3D collagen hydrogels when applied at the appropriate concentration. Moreover, collagen hydrogel constructs containing MSCs and appropriate growth factor stimulus might be a potentially useful biological tool for 3D bone tissue engineering.     

Adhesion and migration of marrow-derived osteoblasts on injectable in situ crosslinkable poly(propylene fumarate-co-ethylene glycol)-based hydrogels with a covalently linked RGDS peptide

Marrow-derived osteoblasts were cultured on poly(propylene fumarate-co-ethylene glycol) (P(PF-co-EG)) based hydrogels modified in bulk with a covalently linked RGDS model peptide. A poly(ethylene glycol) spacer arm was utilized to covalently link the peptide to the hydrogel. Three P(PF-co-EG) block copolymers were synthesized with varying poly(ethylene glycol) block lengths relative to poly(ethylene glycol) spacer arm. A poly(ethylene glycol) block length of nominal molecular weight 2000 and spacer arm of nominal molecular weight 3400 were found to reduce nonspecific cell adhesion and show RGDS concentration dependent marrow-derived osteoblast adhesion. A concentration of 100 nmol/mL RGDS was sufficient to promote adhesion of 84 +/- 17% of the initial seeded marrow-derived osteoblasts compared with 9 +/- 1% for the unmodified hydrogel after 12 h. Cell spreading was quantified as a method for evaluating adhesivity of cells to the hydrogel. A megacolony migration assay was utilized to assess the migration characteristics of the marrow-derived osteoblasts on RGDS modified hydrogels. Marrow-stromal osteoblasts migration was greater on hydrogels modified with 100 nmol/mL linked RGDS when compared with hydrogels modified with 1000 nmol/mL linked RGDS, while proliferation was not affected. These P(PF-co-EG) hydrogels modified in the bulk with RGDS peptide are potential candidates as in situ forming scaffolds for bone tissue engineering applications.     

The effect of mesenchymal stem cell osteoblastic differentiation on the mechanical properties of engineered bone-like tissue

Mesenchymal stem cells (MSCs) can give rise to osteoblasts and have therefore been suggested as a cell source for bone engineering. Here we hypothesized that MSC osteoblastic differentiation and maturation can be supported by three-dimensional cultures in collagen hydrogels (hydrogel culture) to ultimately give rise to mechanically robust bone-like tissue. We first compared the osteoblastic differentiation efficiency of MSCs using osteoinductive supplements (β-glycerophosphate, vitamin C, and dexamethasone) in a hydrogel culture and in a two-dimensional culture (2D culture) by assessing surrogate parameters for osteoblastic differentiation, including osteocalcin (OC) secretion and calcium (Ca) deposition. We next constructed ring-shaped bone-like tissues using MSCs in the hydrogel cultures, and assessed their mechanical (strain-strain analysis), biochemical/molecular (OC secretion, Ca deposition, and Runx2/osterix mRNA levels), and morphological (von Kossa staining) properties. OC secretions and Ca depositions were significantly higher in the hydrogel cultures than those in the 2D cultures, suggesting better osteoblastic differentiation and maturation in the hydrogel cultures. Collagen hydrogel-based ring-shaped bone-like tissues conditioned with osteoinductive supplements developed enhanced biomechanical properties, including high tissue stiffness and ultimate burst strength, superior molecular/biochemical properties, and morphological signs typically found in mineralized bone. These results may be exploited not only to generate bioartificial bone, but also to elucidate the basic mechanisms of bone physiology.     

Maleimide-thiol coupling of a bioactive peptide to an elastin-like protein polymer

Recombinant elastin-like protein (ELP) polymers display several favorable characteristics for tissue repair and replacement as well as drug delivery applications. However, these materials are derived from peptide sequences that do not lend themselves to cell adhesion, migration, or proliferation. This report describes the chemoselective ligation of peptide linkers bearing the bioactive RGD sequence to the surface of ELP hydrogels. Initially, cystamine is conjugated to ELP, followed by the temperature-driven formation of elastomeric ELP hydrogels. Cystamine reduction produces reactive thiols that are coupled to the RGD peptide linker via a terminal maleimide group. Investigations into the behavior of endothelial cells and mesenchymal stem cells on the RGD-modified ELP hydrogel surface reveal significantly enhanced attachment, spreading, migration and proliferation. Attached endothelial cells display a quiescent phenotype.     

Attachment and spreading of fibroblasts on an RGD peptide-modified injectable hyaluronan hydrogel

Hyaluronan (HA) hydrogels resist attachment and spreading of fibroblasts and most other mammalian cell types. A thiol-modified HA (3,3'-dithiobis(propanoic dihydrazide) [HA-DTPH]) was modified with peptides containing the Arg-Gly-Asp (RGD) sequence and then crosslinked with polyethylene glycol (PEG) diacrylate (PEGDA) to create a biomaterial that supported cell attachment, spreading, and proliferation. The hydrogels were evaluated in vitro and in vivo in three assay systems. First, the behavior of human and murine fibroblasts on the surface of the hydrogels was evaluated. The concentration and structure of the RGD peptides and the length of the PEG spacer influenced cell attachment and spreading. Second, murine fibroblasts were seeded into HA-DTPH solutions and encapsulated via in situ crosslinking with or without bound RGD peptides. Cells remained viable and proliferated within the hydrogel for 15 days in vitro. Although the RGD peptides significantly enhanced cell proliferation on the hydrogel surface, the cell proliferation inside the hydrogel in vitro was increased only modestly. Third, HA-DTPH/PEGDA/peptide hydrogels were evaluated as injectable tissue engineering materials in vivo. A suspension of murine fibroblasts in HA-DTPH was crosslinked using PEGDA plus PEGDA peptide, and the viscous, gelling mixture was injected subcutaneously into the flanks of nude mice; gels formed in vivo following injection. After 4 weeks, growth of new fibrous tissue had been accelerated by the sense RGD peptides. Thus, attachment, spreading, and proliferation of cells is dramatically enhanced on RGD-modified surfaces but only modestly accelerated in vivo tissue formation.     

In vitro osteogenic differentiation of marrow stromal cells encapsulated in biodegradable hydrogels

Novel hydrogel materials based on oligo(poly(ethylene glycol) fumarate) (OPF) crosslinked with a redox radical initiation system were recently developed in our laboratory as injectable cell carriers for orthopedic tissue engineering applications. The effect of OPF hydrogel material properties on in vitro osteogenic differentiation of encapsulated rat marrow stromal cells (MSCs) with and without the presence of osteogenic supplements (dexamethasone) was investigated. Two OPF formulations that resulted in hydrogels with different swelling properties were used to encapsulate rat MSCs (seeding density approximately 13 million cells/mL, samples 6 mm diameter x 0.5 mm thick before swelling) and osteogenic differentiation in these constructs over 28 days in vitro was determined via histology and biochemical assays for alkaline phosphatase, osteopontin and calcium. Evidence of MSC differentiation was apparent over the culture period for samples without dexamethasone, but there was large variability in calcium production between constructs using cells of the same source. Differentiation was also seen in samples cultured with osteogenic supplements, but calcium deposition varied depending on the source pool of MSCs. By day 28, osteopontin and calcium results suggested that, in the presence of dexamethasone, OPF hydrogels with greater swelling promoted embedded MSC differentiation over those that swelled less (43.7 +/- 16.5 microg calcium/sample and 16.4 +/- 2.8 microg calcium/sample, respectively). In histological sections, mineralized areas were apparent in all sample types many microns away from the cells. These experiments indicate that OPF hydrogels are promising materials for use as injectable MSC carriers and that hydrogel swelling properties can influence osteogenic differentiation of encapsulated progenitor cells.     

Enhanced chondrogenesis of mesenchymal stem cells in collagen mimetic peptide-mediated microenvironment

A new type of synthetic hydrogel scaffold that mimics certain aspects of structure and function of natural extracellular matrix (ECM) has been developed. We previously reported the conjugation of collagen mimetic peptide (CMP) to poly(ethylene oxide) diacrylate (PEODA) to create a polymer-peptide hybrid scaffold for a suitable cell microenvironment. In this study, we showed that the CMP-mediated microenvironment enhances the chondrogenic differentiation of mesenchymal stem cells (MSCs). MSCs were harvested and photo-encapsulated in CMP-conjugated PEODA (CMP/PEODA). After 3 weeks, the histological and biochemical analysis of the CMP/PEODA gel revealed twice as much glycosaminoglycan and collagen contents as in control PEODA hydrogels. Moreover, MSCs cultured in CMP/PEODA hydrogel exhibited a lower level of hypertrophic markers, core binding factor alpha 1, and type X collagen than MSCs in PEODA hydrogel as revealed by gene expression and immunohistochemisty. These results indicate that CMP/PEODA hydrogel provides a favorable microenvironment for encapsulated MSCs and regulates their downstream chondrogenic differentiation.     

Flow perfusion culture induces the osteoblastic differentiation of marrow stroma cell-scaffold constructs in the absence of dexamethasone

Flow perfusion culture of scaffold/cell constructs has been shown to enhance the osteoblastic differentiation of rat bone marrow stroma cells (MSCs) over static culture in the presence of osteogenic supplements including dexamethasone. Although dexamethasone is known to be a powerful induction agent of osteoblast differentiation in MSC, we hypothesied that the mechanical shear force caused by fluid flow in a flow perfusion bioreactor would be sufficient to induce osteoblast differentiation in the absence of dexamethasone. In this study, we examined the ability of MSCs seeded on titanium fiber mesh scaffolds to differentiate into osteoblasts in a flow perfusion bioreactor in both the presence and absence of dexamethasone. Scaffold/cell constructs were cultured for 8 or 16 days and osteoblastic differentiation was determined by analyzing the constructs for cellularity, alkaline phosphatase activity, and calcium content as well as media samples for osteopontin. For scaffold/cell constructs cultured under flow perfusion, there was greater scaffold cellularity, alkaline phosphatase activity, osteopontin secretion, and calcium deposition compared with static controls, even in the absence of dexamethasone. When dexamethasone was present in the cell culture medium under flow perfusion conditions, there was further enhancement of osteogenic differentiation as evidenced by lower scaffold cellularity, greater osteopontin secretion, and greater calcium deposition. These results suggest that flow perfusion culture alone induces osteogenic differentiation of rat MSCs and that there is a synergistic effect of enhanced osteogenic differentiation when both dexamethasone and flow perfusion culture are used.     

Enzymatic conjugation of a bioactive peptide into an injectable hyaluronic acid–tyramine hydrogel system to promote the formation of functional vasculature

In this study, one-step enzyme-mediated preparation of a multi-functional injectable hyaluronic-acid-based hydrogel system is reported. Hydrogel was formed through the in situ coupling of phenol moieties by horseradish peroxidase (HRP) and hydrogen peroxide (H₂O₂), and bioactive peptides were simultaneously conjugated into the hydrogel during the gel formation process. The preparation of this multi-functional hydrogel was made possible by synthesizing peptides containing phenols which could couple with the phenol moieties of hyaluronic-acid–tyramine (HA–Tyr) during the HRP-mediated crosslinking reaction. Preliminary studies demonstrated that two phenol moieties per molecule resulted in a consistently high degree of conjugation into the HA–Tyr hydrogel network, unlike the one modified with one phenol moiety per molecule. Therefore, an Arg–Gly–Asp (RGD) peptide bearing two phenol moieties (phenol₂–poly(ethylene glycol)–RGD) was designed for conjugation to endow the HA–Tyr hydrogel with adhesion signals and enhance its bioactivities. Human umbilical vein endothelial cells (HUVECs) cultured on or within the RGD-modified hydrogels showed significantly different adhesion behavior, from non-adherence on the HA–Tyr hydrogel to strong adhesion on hydrogels modified with phenol₂–poly(ethylene glycol)–RGD. This altered cell adhesion behavior led to improved cell proliferation, migration and formation of capillary-like network in the hydrogel in vitro. More importantly, when HUVECs and human fibroblasts (HFF1) were encapsulated together in the RGD-modified HA–Tyr hydrogel, functional vasculature was observed inside the cell-laden gel after 2 weeks in the subcutaneous tissue. Taken together, the in situ conjugation of phenol₂–poly(ethylene glycol)–RGD into HA–Tyr hydrogel system, coupled with the ease of incorporating cells, offers a simple and effective means to introduce biological signals for preparation of multi-functional injectable hydrogels for tissue engineering application.     

The effects of heparin releasing hydrogels on vascular smooth muscle cell phenotype

Poly(ethylene glycol) diacrylate (PEGDA) hydrogel scaffolds were engineered to promote contractile smooth muscle cell (SMC) phenotype via controlled release of heparin. The scaffold design was evaluated by quantifying the effects of free heparin on SMC phenotype, engineering hydrogels to provide controlled release of heparin, and synthesizing cell-adhesive, heparin releasing hydrogels to promote contractile SMC phenotype. Heparin inhibited SMC proliferation and up-regulated expression of contractile SMC phenotype markers, including smooth muscle α-actin, calponin, and SM-22α, in a dose-dependent fashion (6 μg/ml to 3.2 mg/ml). Heparin release from PEGDA hydrogels was controlled by altering PEGDA molecular weight (MW 1000–6000) and concentration at polymerization (10–30% w/w), yielding release profiles ranging from hours to weeks in duration. Heparin released from PEGDA gels, formulated for optimized heparin loading and release kinetics (30% w/w PEGDA, MW 3000), stimulated SMCs to up-regulate contractile marker mRNA. A cell-instructive scaffold construct was prepared by polymerizing a thin hydrogel film, with pendant RGD peptides for cell attachment, over the optimized hydrogel depots. SMCs seeded on these constructs had elevated levels of contractile marker mRNA after 3 d of culture compared with SMCs on control constructs. These results indicate that RGD-modified, heparin releasing PEGDA gels can act as cell-instructive scaffolds that promote contractile SMC phenotype.     

Inhibition of in vitro chondrogenesis in RGD-modified three-dimensional alginate gels

The goal of this study was to investigate the effects of adhesion to the arginine-glycine-aspartic acid (RGD) sequence on the chondrogenesis of bone marrow stromal cells (BMSCs). Synthetic RGE- and RGD-containing peptides were conjugated to sodium alginate, and bovine BMSCs were seeded onto 2D alginate surfaces or encapsulated in 3D gels. BMSCs spread specifically on RGD-modified surfaces, and spreading was inhibited by a soluble RGD peptide and by anti-beta1 and anti-alpha(v)beta3 integrin blocking antibodies. After 7 days in 3D gel culture, the chondrogenic supplements (TGF-beta1 and dexamethasone) significantly stimulated chondrocytic gene expression (collagen II, aggrecan, and Sox-9) and matrix accumulation (collagen II and sGAG) in RGE-modified gels, but this response was inhibited in the RGD-modified gels. Inhibition of sGAG synthesis increased with increasing RGD density, and synthesis was partially rescued by adding a soluble RGD peptide. Addition of an anti-alpha(v)beta3 integrin blocking antibody had no effect on chondrogenesis, while an anti-alpha5 antibody reduced sGAG accumulation. Overall, this study demonstrates that interaction with the RGD motif significantly inhibits the initial chondrogenesis of BMSCs within 3D alginate gels. These results provide new insights into the role of cell-matrix interactions in regulating chondrogenesis and highlight the importance of choosing appropriate biomaterials for tissue engineering therapies.     

In vitro generation of an osteochondral construct using injectable hydrogel composites encapsulating rabbit marrow mesenchymal stem cells

Injectable, biodegradable hydrogel composites of crosslinked oligo(poly(ethylene glycol) fumarate) (OPF) and gelatin microparticles (MPs) were utilized to fabricate a bilayered osteochondral construct consisting of a chondrogenic layer and an osteogenic layer, and to investigate the differentiation of rabbit marrow mesenchymal stem cells (MSCs) encapsulated in both layers in vitro. The results showed that MSCs in the chondrogenic layer were able to undergo chondrogenic differentiation, especially in the presence of TGF-β1-loaded MPs. In the osteogenic layer, cells maintained their osteoblastic phenotype. Although calcium deposition in the osteogenic layer was limited, cells in the osteogenic layer significantly enhanced chondrogenic differentiation of MSCs in the chondrogenic layer. The greatest effect was observed when MSCs were encapsulated with TGF-β1-loaded MPs and cultured with osteogenic cells in the bilayered constructs. Overall, this study demonstrates the fabrication of bilayered hydrogel composites that mimic the structure and function of osteochondral tissue, along with the application of these composites as cell and growth factor carriers.     

Human mesenchymal stem cell differentiation to NP-like cells in chitosan-glycerophosphate hydrogels

Intervertebral disc (IVD) degeneration is one of the major causes of low back pain. As current clinical treatments are aimed at restoring biomechanical function and providing symptomatic relief, interest in methods focused on biological repair has increased. Several tissue engineering approaches using different cell types and hydrogels/scaffolds have been proposed. Owing to the unsuitable nature of degenerate cells for tissue engineering attention has focused on the use of mesenchymal stem cells (MSCs). Additionally, while rigid scaffolds have been demonstrated to allow MSC differentiation to the chondrocyte-like cells of the IVD, hydrogels are being increasingly studied as they allow minimally invasive implantation without extensive damage to the IVD. Here, we have studied the temperature-sensitive hydrogel chitosan-glycerophosphate (C/Gp), seeded with human MSCs and cultured for 4 weeks in standard medium. We have analysed the gene and protein expression profile of the MSCs and compared it to that of both nucleus pulposus (NP) cells and articular chondrocytes cultured in C/Gp. Gene expression analysis for chondrocytic-cell marker genes demonstrated differentiation of MSCs to a phenotype which showed similarities to both articular chondrocytes and NP cells. Conventional PCR demonstrated a lack of expression of osteogenic marker genes and the hypertrophic marker gene type X collagen. MSCs also secreted both proteoglycans and collagens in a ratio, which more closely resembled that of NP cells than articular chondrocytes. These results therefore suggest that MSC-seeded C/Gp gels could be used clinically for the regeneration of the degenerate human IVD.     

Biofabricated marine hydrozoan: a bioactive crystalline material promoting ossification of mesenchymal stem cells

This study introduces a novel three-dimensional biomatrix obtained from the marine hydrocoral Millepora dichotoma as a scaffold for hard tissue engineering. Millepora dichotoma was biofabricated under field and laboratory conditions. Three-dimensional biomatrices were made in order to convert mesenchymal stem cells (MSCs) to exemplify osteoblastic phenotype. We investigated the effect of the biomatrices on MSCs proliferation and differentiation at 2, 3, 4, 7, 10, 14, 21, 28, and 42 days. Different analyses were made: light microscopy, scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS), calcium incorporation to newly formed tissue (alizarin red), bone nodule formation (von Kossa), fat aggregate formation (oil red O), collagen type I immunofluorescence, DNA concentrations, alkaline phosphatase (ALP) activity, and osteocalcin concentrations. MSCs seeded on Millepora dichotoma biomatrices showed higher levels of calcium and phosphate incorporation and higher type I collagen levels than did control Porites lutea biomatrices. ALP activity revealed that MSCs seeded on M. dichotoma biomatrices are highly osteogenic compared to those on control biomatrices. The osteocalcin content of MSCs seeded on M. dichotoma remained constant up to 2 weeks before rising to surpass that of seeded P. lutea biomatrices after 28 days. Our study thus showed that M. dichotoma biomatrices enhance the differentiation of MSCs into osteoblast and hence have excellent potential as bioscaffold for hard tissue engineering.