sepharose and alizarin

Dentin sialophosphoprotein (DSPP) has been shown to play a primary role in the formation and growth of hydroxyapatite crystals in an extracellular matrix of hard tissue such as bone and teeth. We hypothesized that the mineralization ability of DSPP might depend on a specific domain within it. Three peptides, which have hydroxyapatite (HA) binding affinity, denoted as mineralization inducing peptide (MIP1, MIP2, and MIP3) were identified from DSPP. The both of MIP2 and MIP3 had HA nucleation activity demonstrated by XRD. Among three MIPs, MIP3 significantly supported the human bone marrow stromal cell differentiation into osteoblastic cells. An immunoblot with antibodies specific for the phosphorylated forms of ERK was conducted with cells treated by MIP3. MIP3 transduced intracellular signals via the ERK pathways and was able to induce osteoblastic differentiation, as seen by high expression of ALP, type 1 collagen, OC, OPN, and Runx2 in accordance with applied MIP3 concentration. The Asp, Glu, and Ser residues in MIP3 play important roles for the affinity of calcium in HA bone mineral. Further animal experiment with MIP3 in combination with hydroxyapatite mineral induced marked new bone formation for 4 weeks at rabbit calvarial defect model. The new bone area was much higher in test group, implying that the peptide modified group had excellent biocompatibility when compared with the unmodified group. Taken together, the MIP from DSPP has potential to enhance mineralization followed by to enhance osteoblastic differentiation and bone regeneration.

Topics: Amino Acid Sequence; Animals; Anthraquinones; Bone Regeneration; Calcification, Physiologic; Cell Differentiation; Durapatite; Extracellular Matrix Proteins; Gene Expression Regulation; Humans; Implants, Experimental; Male; Molecular Sequence Data; Osteoblasts; Osteogenesis; Peptides; Phosphoproteins; Rabbits; Sepharose; Sialoglycoproteins; Signal Transduction; Skull

The development of osteochondral tissue engineered interfaces would be a novel treatment for traumatic injuries and aging associated diseases that affect joints. This study reports the development of a bilayered scaffold, which consists of both bone and cartilage regions. On the other hand, amniotic fluid-derived stem cells (AFSCs) could be differentiated into either osteogenic or chondrogenic cells, respectively. In this study we have developed a bilayered scaffolding system, which includes a starch/polycaprolactone (SPCL) scaffold for osteogenesis and an agarose hydrogel for chondrogenesis. AFSC-seeded scaffolds were cultured for 1 or 2 weeks in an osteochondral-defined culture medium containing both osteogenic and chondrogenic differentiation factors. Additionally, the effect of the presence or absence of insulin-like growth factor-1 (IGF-1) in the culture medium was assessed. Cell viability and phenotypic expression were assessed within the constructs in order to determine the influence of the osteochondral differentiation medium. The results indicated that, after osteogenic differentiation, AFSCs that had been seeded onto SPCL scaffolds did not require osteochondral medium to maintain their phenotype, and they produced a protein-rich, mineralized extracellular matrix (ECM) for up to 2 weeks. However, AFSCs differentiated into chondrocyte-like cells appeared to require osteochondral medium, but not IGF-1, to synthesize ECM proteins and maintain the chondrogenic phenotype. Thus, although IGF-1 was not essential for creating osteochondral constructs with AFSCs in this study, the osteochondral supplements used appear to be important to generate cartilage in long-term tissue engineering approaches for osteochondral interfaces. In addition, constructs generated from agarose-SPCL bilayered scaffolds containing pre-differentiated AFSCs may be useful for potential applications in regeneration strategies for damaged or diseased joints.

Topics: Aggrecans; Alkaline Phosphatase; Amniotic Fluid; Anthraquinones; Cell Differentiation; Cell Survival; Cells, Immobilized; Chondrogenesis; Collagen Type II; Core Binding Factor Alpha 1 Subunit; Culture Media; Fluorescent Antibody Technique; Humans; Microscopy, Electron, Scanning; Osteogenesis; Polyesters; Sepharose; Spectrometry, X-Ray Emission; Staining and Labeling; Starch; Stem Cells; Tissue Scaffolds