Chondrogenesis from human placenta-derived mesenchymal stem cells in three-dimensional scaffolds for cartilage tissue engineering.

Human placenta-derived mesenchymal stem cells (hPMSCs) represent a promising source of stem cells. The application of hPMSCs in cartilage tissue engineering, however, was less reported. In this study, hPMSCs were grown in a three-dimensional (3D) environment for cartilage tissue formation in vitro. To select proper scaffolds for 3D culture of mesenchymal stem cells (MSCs), rat adipose-derived MSCs were initially employed to optimize the composition and condition of the 3D environment. The suitability of a poly(D,L-lactide-co-glycolide) (PLGA) precision scaffold previously developed for seeding and culture of primary chondrocytes was tested for MSCs. It was established that MSCs had to be embedded in alginate gel before seeded in the PLGA precision scaffold for cartilage-like tissue formation. The inclusion of nano-sized calcium-deficient hydroxyapatite (nCDHA) and/or a recombinant protein containing arginine-glycine-aspartate (RGD) into the alginate gel enhanced the chondrogenesis for both rat adipose-derived MSCs and hPMSCs. The amount of extracellular matrix such as glycosaminoglycan and type II collagen accumulated during a period of 21 days was found to be the greatest for hPMSCs embedded in the alginate/nCDHA/RGD gel and injected and cultivated in the precision scaffold. Also, histological analyses revealed the lacunae formation and extracellular matrix production from the seeded hPMSCs. Comparing human bone marrow-derived MSCs (hBMSCs) and hPMSCs grown in the previous composite scaffolds, the secretion of glycosaminoglycan was twice as higher for hPMSCs as that for hBMSCs. It was concluded that the alginate/nCDHA/RGD mixed gel in the aforementioned system could provide a 3D environment for the chondrogenesis of hPMSCs, and the PLGA precision scaffold could provide the dimensional stability of the whole construct. This study also suggested that hPMSCs, when grown in a suitable scaffold, may be a good source of stem cells for building up the tissue-engineered cartilage.

Immersion behavior of gelatin-containing calcium phosphate cement.

Calcium phosphate cements (CPCs) have many favorable properties that support their clinical use as bone defect repair. However, it is difficult to deliver to the required site and hard to compact adequately due to inherently low ductility of ceramics. The aim of this study focused on the effect of the gelatin content on properties of CPCs. The diametral tensile strength, morphology, and weight loss of gelatin cements were evaluated after immersion in physiological solution, in addition to setting time. The results indicated that the setting time significantly increased with increasing gelatin amount. The 2 wt.% gelatin could make CPCs attain the maximum strength value of 2.1 MPa at 15-day immersion, while 1.6 MPa for the cement without gelatin. It is concluded that the presence of gelatin improved mechanical properties of CPCs; in particular, 2 wt.% gelatin. CPCs containing 2 wt.% gelatin hardened in an acceptable time recommended for clinical applications.

Artificial extracellular matrix proteins contain heparin-binding and RGD-containing domains to improve osteoblast-like cell attachment and growth.

Through the recombinant DNA technology, it is possible to create artificial extracellular matrix (aECM) proteins with domains chosen to modulate cellular behaviors. In this study, we constructed three aECM proteins containing both heparin-binding and RGD-containing domains (387RGDS, Tri-FN10, and TNC-FN3) produced in Escherichia coli. Promotion of MG-63 cell attachment and growth in two-dimensional (2D) cultures (tissue culture plate, polycaprolactone membrane) and 3D cultures (Cytodex 1 and Plastic Plus microcarriers) were demonstrated on these three aECM protein-coated surfaces. These three aECM proteins improved MG-63 cell attachment and growth in the order TNC-FN3 > Tri-FN10 > 387RGDS in both 2D and 3D cultures. This study is the first report of the construction of aECM proteins that contain both heparin-binding and RGD-containing domains used in osteoblast tissue engineering applications.

Evaluation of biodegradable polyesters modified by type II collagen and Arg-Gly-Asp as tissue engineering scaffolding materials for cartilage regeneration.

Synthetic biodegradable polyesters poly(L-lactide) (PLLA) and poly(D,L-lactide-coglycolide) (PLGA) (50:50) modified by porcine type II collagen and an Arg-Gly-Asp (RGD)-containing protein were evaluated as scaffolds for cartilage regeneration in this study. Cytocompatibility of the polymer films was tested using immortalized chondrocytes. Neocartilage formation in vitro on cell-seeded scaffolds was further examined using primary porcine chondrocytes. The inflammatory response of the scaffolds was evaluated subcutaneously in rats. A pilot animal study was conducted, in which rabbit allogeneic chondrocyte-seeded scaffolds were implanted to repair the defected rabbit knee cartilage. The results demonstrated that PLGA as well as its blends with PLLA had better cell growth than pure PLLA, and that type II collagen enhanced, but RGD inhibited cell proliferation. Scaffolds made of blended PLLA/PLGA had larger dynamic compressive modulus compared to scaffolds made of PLLA or PLGA single polymer. Chondrocyte-seeded scaffolds modified by type II collagen without RGD had the greater number of cells as well as higher glycosaminoglycan (GAG) and collagen contents compared to scaffolds without type II collagen modification or scaffolds further modified with RDG. Type II collagen modification prevented infiltration by host tissue and capsule formation. Unmodified PLLA and PLLA/PLGA constructs demonstrated persisting inflammatory response after 6 months, while all type II collagen-modified PLLA/PLGA constructs showed complete repair and no inflammation. Partial or full repair was observed after 2 months of postimplantation in type II collagen-modified PLLA/PLGA constructs, with equal cellularity and 75-80% matrix contents of a normal rabbit articular cartilage. It was concluded that PLLA/PLGA blended scaffolds modified by type II collagen were a potential tissue engineering scaffold for cartilage regeneration.

Evaluation of chitosan-alginate-hyaluronate complexes modified by an RGD-containing protein as tissue-engineering scaffolds for cartilage regeneration.

In this study, a series of natural biodegradable materials in the form of chitosan (C)-alginate (A)-hyaluronate (H) complexes are evaluated as tissue-engineering scaffolds. The weight ratio of C/A is 1 : 1 or 1 : 2. Sodium hyaluronate is mixed in 2%. The complexes can be cast into films or fabricated as scaffolds. Their surface can be further modified by an Arg-Gly-Asp (RGD)-containing protein, a cellulose-binding domain-RGD (R). Cytocompatibility tests of the films are conducted using immortalized rat chondrocyte (IRC) as well as primary articular chondrocytes harvested from rabbits. The neocartilage formation in cell-seeded scaffolds is examined in vitro as well as in rabbits, where the scaffolds are implanted into the defect-containing joints. The results from cytocompatibility tests demonstrate that R enhances cell attachment and proliferation on C-A and C-A-H complex films. Complex C1A1HR (C : A = 1 : 1 with H and R) has better performance than the other formulation. Cells retain their spherical morphology on all C-A and C-A-H complexes. The in vitro evaluation of the seeded scaffolds indicates that the C1A1HR complex is the most appropriate for 3-D culture, manifested by the better cell growth as well as higher glycosaminoglycan and collagen contents. When the chondrocyte scaffolds are implanted into rabbit knee cartilage defects, partial repair is observed after 1 month in C1A1HR as well as in C1A1 (C : A = 1 : 1 without H and R) scaffolds. The defects are completely repaired in 6 months when C1A1HR constructs are implanted. It is concluded that C1A1HR is a potential tissue-engineering scaffold for cartilage regeneration.

The effect of an RGD-containing fusion protein CBD-RGD in promoting cellular adhesion.

The effect of a recombinant RGD (arginine-glycine-aspartic acid)-containing fusion protein, cellulose-binding domain (CBD)-RGD, on the cellular adhesion to a biomedical polyurethane (PU) was evaluated. A series of different cell lines, as well as freshly harvested animal cells, were grown on the PU surfaces with or without CBD-RGD, in serum or serum-free media. The results showed that the enhancement of cellular attachment by CBD-RGD varied with cell types. This is believed to be a result of the unique integrin receptors on each type of cell surface. The existence of certain divalent ions (Mg2+ and Mn2+) may increase the efficacy of the CBD-RGD, in a cell type-dependent manner. The fusion protein was also found to inhibit the platelet activation. The effect of CBD-RGD was further examined in two other substrate materials, poly(L-lactide) (PLLA) and poly(lactide-co-glycolide) (PLGA). The effect on cellular adhesion correlated with the amount of CBD-RGD physically adsorbed on the material surface.

Improved retention of endothelial cells seeded on polyurethane small-diameter vascular grafts modified by a recombinant RGD-containing protein.

Sponge-type small-diameter vascular grafts were fabricated from a medical-grade polyurethane, Pellethane 2363-80A, by utilization of a salt casting technique. The grafts were compliance matched with a storage modulus of 0.53 +/- 0.08 MPa. The luminal surface of grafts was modified with a thin layer ( approximately 40 micro m) of gelatin crosslinked by epoxide. Then a special Arg-Gly-Asp (RGD)-containing recombinant protein, named CBD-RGD (cellulose binding domain RGD-containing protein), was coated onto the gelatin layer. The platelet adhesion and activation on such a gelatin/CBD-RGD modified surface was significantly reduced. Human umbilical vein endothelial cells were seeded more efficiently onto the modified grafts. There was also a substantial reduction in the subsequent loss of cells from the graft surface following perfusion in vitro. The cell number retained on the modified graft was enhanced by three times after 1 h of perfusion, and by eight times after 3 h of perfusion (retention rate approximately 63%). The retention after 3 h of perfusion could be further increased to nearly 100% if the lined endothelium on gelatin/CBD-RGD modified graft was cultured for another week before perfusion. The modified surface was also shown to help canine external jugular vein endothelial cells to maintain the round cell morphology in vitro.