https://www.seas.upenn.edu/%7Edischer/pdfs/Cell_on_Gel-CELLpaper.pdf

Matrix elasticity directs stem cell lineage specification.

Fibroblasts can condense a hydrated collagen lattice to a tissue-like structure 1/28th the area of the starting gel in 24 hr. The rate of the process can be regulated by varying the protein content of the lattice, the cell number, or the con- centration of an inhibitor such as Colcemid. Fibroblasts of high population doubling level propagated in vitro, which have left the cell cycle, can carry out the contraction at least as efficiently as cycling cells. The potential uses of the system as an immu- nologically tolerated "tissue" for wound hea ing and as a model for studying fibroblast function are discussed.

Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative

http://www.ibmc.up.pt/projectos/FJMonteiro/Researchers/MJCoelho/Coelho%20MJ%203.pdf

Human bone cell cultures in biocompatibility testing. Part II : effect of ascorbic acid, β-glycerophosphate and dexamethasone on osteoblastic differentiation

Effect of bone extracellular matrix synthesized in vitro on the osteoblastic differentiation of marrow stromal cells

A novel approach was utilized to grow in vitro mineralized bone tissue using lighter-than-water, polymeric scaffolds in a high aspect ratio rotating bioreactor. We have adapted polymer microencapsulation methods for the formation of hollow, lighter-than-water microcarriers of degradable poly(lactic-co-glycolic acid). Scaffolds were fabricated by sintering together lighter-than-water microcarriers from 500 to 860 μm in diameter to create a fully interconnected, three-dimensional network with an average pore size of 187 μm and aggregate density of 0.65 g/mL. Motion in the rotating bioreactor was characterized by numerical simulation and by direct measurement using an in situ particle tracking system. Scaffold constructs established a near circular trajectory in the fluid medium with a terminal velocity of 98 mm/s while avoiding collision with the bioreactor wall. Preliminary cell culture studies on these scaffolds show that osteoblast-like cells readily attached to microcarrier scaffolds using controlled seeding conditions with an average cell density of 6.5 × 104 cells/cm2. The maximum shear stress imparted to attached cells was estimated to be 3.9 dynes/cm2. In addition, cells cultured in vitro on these lighter-than-water scaffolds retained their osteoblastic phenotype and showed significant increases in alkaline phosphatase expression and alizarin red staining by day 7 as compared with statically cultured controls. © 2001 John Wiley & Sons, Inc. J Biomed Mater Res 55: 242–253, 2001

Bone tissue engineering in a rotating bioreactor using a microcarrier matrix system.

Adult human osteoblastic cells were grown in a native type I collagen gel. Proliferation and viability analyses showed that cells rapidly stopped dividing and became blocked in the G0G1 phase (91% on day 13). Carboxyfluorescein diacetate cell staining and flow cytometry showed that osteoblasts were viable for the first 16 days and then viability decreased (58% viable cells on day 22). Osteoblasts were able to retract the matrix. Betaglycerophosphate (βGP) stimulated the deposition of mineral particles in the collagen network, and electron probe microanalysis showed that they were principally calcium and phosphorus, with a Ca/P ratio of about 1.7. Various times of βGP supply were tested. We compared 10 mM βGP added only once at day 0, or continuously from day 0, day 8, or day 21. Mineralization was observed in conditions where βGP was added at day 0. Furthermore, 10 mM βGP added once during gel preparation was sufficient to induce mineralization with mineral accumulation up to day 15 whereas the speed of the gel contraction decreased. In every condition, cultures expressed high alkaline phosphatase (ALP) levels as early as day 3, which decreased afterwards. These kinetics might explain why the other conditions did not prove favorable to the mineralization process. The model was used to study the influence of blocking gel retraction. Blocking retraction delayed the ALP activity decrease, but had no effect on mineralization. In conclusion, human adult osteoblasts cultured in native collagen gel stopped proliferation and underwent mineralization very early. This model should be used to investigate the influence of effectors on the early stages of culture.

Mineralization and alkaline phosphatase activity in collagen lattices populated by human osteoblasts.

Human osteoblastic cells were isolated enzymatically from adult human spongy bone and grown in MEM-Ham F12 1:1 medium supplemented with 2% Ultroser (USM). They were subcultured and examined for osteoblast features by morphological, histological, and biochemical approaches. The cells had a characteristic polyhedral morphology and produced a high level of alkaline phosphatase (ALKP). Confluent cultures were uniformly stained for ALKP and flow cytometry analysis with fluorescein diphosphate gave a single peak signal, reflecting a highly positive population, distinct from cultures of fibroblasts. The ALKP activity was stimulated by 1,25 (OH)2 vitamin D3. CD 44 was strongly expressed in these cultures, although osteoblasts are negative in vivo and osteocytes are positive. The main collagen synthesized was type I collagen and osteocalcin was produced after stimulation by vitamin D3. 10 mM βGP induced mineralization and microprobe analysis of the crystals showed a composition close to hydroxyapatite.

O. Sabido

Papers

Mineralization and alkaline phosphatase activity in collagen lattices populated by human osteoblasts.

Characterization of human osteoblastic cells: influence of the culture conditions