Monday, August 15, 2011

growing tall with interleukins

In vitro engineering of cartilage: effects of serum substitutes, TGF-beta, and IL-1alpha.

"We used porous collagen sponges to assess the effects of serum substitutes and exogenous TGF-beta1 and IL-1alpha on chondrocytes (bovine articular chondrocytes, bACs) and on chondroinduced human dermal fibroblasts (hDFs). We determined the effects of low concentrations of FBS[fetal bovine serum] and two serum substitutes, Nutridoma and ITS(+3), on cellularity and matrix production. After culture for intervals, sponges were harvested for histological and biochemical measurement of cartilage-specific chondroitin 4-sulfate proteoglycan (C 4-S PG).
Cultured bACs showed equivalent growth in Nutridoma (1%) and 10% FBS. Both TGF-beta1 and IL-1alpha significantly stimulated accumulation of C 4-S PG by bACs in 3D porous collagen sponges. Many endogenous growth factors were upregulated in hDFs cultured with chondroinductive DBP. Addition of TGF-beta1 and IL-1alpha for 11 days significantly stimulated accumulation of C 4-S PG by hDFs cultured in DMEM with 1% Nutridoma.
Porous collagen sponges are supportive of chondrogenesis and of chondroinduction by DBP. Optimization of serum-free culture conditions, including growth factors, matrix components, and mechanical stimuli will expedite translation to wider clinical applications. Use of autogenous dermal fibroblasts pre-cultured with DBP and induced to chondrocytes offers an alternative to autogenous chondrocytes."

"In the samples treated with 54 nM IL-1α, the metachromatic matrix around the cells and between the particles of DBP appeared denser and more granular than in samples that were not treated with IL-1α."

" IL-1α is an endogenous, autocrine factor in human osteoblasts: first, osteoblast conditioned medium is a mitogen for other osteoblasts even in the presence of indomethacin but only partially in the presence of IL-1α-neutraling antibody; second, that osteoblasts secrete IL-1α; and, third, that exogenous recombinant human IL-1α stimulated proliferation of normal human osteoblasts"

This study provides contradictory results

Catabolic factors and osteoarthritis-conditioned medium inhibit chondrogenesis of human mesenchymal stem cells.

"We investigated the effect of a catabolic environment on chondrogenesis in pellet cultures of human mesenchymal stem cells (hMSCs). We exposed chondrogenically differentiated hMSC pellets, to interleukin (IL)-1α, tumor necrosis factor (TNF)-α or conditioned medium derived from osteoarthritic synovium (CM-OAS). IL-1α and TNF-α in CM-OAS were blocked with IL-1Ra or Enbrel, respectively. Chondrogenesis was determined by chondrogenic markers collagen type II, aggrecan, and the hypertrophy marker collagen type X on mRNA. Proteoglycan deposition was analyzed by safranin o staining on histology. IL-1α and TNF-α dose-dependently inhibited chondrogenesis when added at onset or during progression of differentiation, IL-1α being more potent than TNF-α. CM-OAS inhibited chondrogenesis on mRNA and protein level but varied in extent between patients. Inhibition of IL-1α partially overcame the inhibitory effect of the CM-OAS on chondrogenesis whereas the TNF-α contribution was negligible. We show that hMSC chondrogenesis is blocked by either IL-1α or TNF-α alone, but that there are additional factors present in CM-OAS that contribute to inhibition of chondrogenesis"

"Addition of IL-1α at day 0, 3, or 7 resulted in a total block of the chondrogenic differentiation as no increased expression of type II collagen and aggrecan mRNA was detected"

The role of IL-6 in bone marrow (BM)-derived mesenchymal stem cells (MSCs) proliferation and chondrogenesis.

"we isolate MSCs from the murine bone marrow, and induce MSCs chondrogenesis with different concentrations of IL-6{up} in vitro.  IL-6 inhibited the differentiation of MSCs into chondrocytes in the dose-dependence manner."

Couldn't get full study.

The inhibition by interleukin 1 of MSC chondrogenesis and the development of biomechanical properties in biomimetic 3D woven PCL scaffolds.

"a three-dimensionally (3D) woven poly(ε-caprolactone) (PCL) scaffold [was] seeded with bone marrow-derived mesenchymal stem cells (MSCs). Pro-inflammatory cytokine interleukin-1 (IL-1) [was present], which is found at high levels in injured or diseased joints. MSC-seeded 3D woven scaffolds cultured in chondrogenic conditions synthesized a functional ECM rich in collagen and proteoglycan content, reaching an aggregate modulus of ~0.75 MPa within 14 days of culture. However, the presence of pathophysiologically relevant levels of IL-1 limited matrix accumulation and inhibited any increase in mechanical properties over baseline values. On the other hand, the mechanical properties of constructs cultured in chondrogenic conditions for 4 weeks prior to IL-1 exposure were protected from deleterious effects of the cytokine. IL-1 significantly inhibits the chondrogenic development and maturation of MSC-seeded constructs; however, the overall mechanical functionality of the engineered tissue can be preserved through the use of a 3D woven scaffold designed to recreate the mechanical properties of native articular cartilage."

"Chondrogenic culture conditions resulted in a reduction in apparent hydraulic permeability of 3 orders of magnitude to approximately 0.0008 mm4/N s"

"Chondrogenic culture conditions containing TGF-β3 resulted in a dense ECM rich in both s-GAGs (as stained by Safranin-O or chondroitin-4-sulfate) and collagens (as stained by Fast Green) that accumulated and completely filled the interstitial voids within the scaffold."

"IL-1 appears to act through the activation of nuclear factor kappa B (NF-κB), as inhibition of this pathway using a dominant-negative suppressor of I-κB, curcumin, or resveratrol abrogates the effects of IL-1 on MSCs."

"relatively high concentrations (100 ng/ml) of bone morphogenetic proteins (BMPs) such as BMP-2 or BMP-9 can partially overcome the effects of IL-1"

"MSCs may play an anti-inflammatory role when reintroduced in vivo and enhance repair by producing IL-1 receptor antagonist (IL-1Ra)"

Interleukin-1β modulates endochondral ossification by human adult bone marrow stromal cells.

"Low doses of IL-1β (50 pg/mL) enhanced colony-forming units-fibroblastic (CFU-f) and -osteoblastic (CFU-o) number (up to 1.5-fold) and size (1.2-fold) in the absence of further supplements and glycosaminoglycan accumulation (1.4-fold) upon BM-MSC chondrogenic induction. In osteogenically cultured BM-MSC, IL-1β enhanced calcium deposition (62.2-fold) and BMP-2 mRNA expression by differential activation of NF-κB and ERK signalling. IL-1β-treatment of BM-MSC generated cartilage resulted in higher production of MMP-13 (14.0-fold) in vitro, mirrored by an increased accumulation of the cryptic cleaved fragment of aggrecan, and more efficient cartilage remodelling/resorption after 5 weeks in vivo (i.e., more TRAP positive cells and bone marrow, less cartilaginous areas), resulting in the formation of mature bone and bone marrow after 12 weeks."

But it is also mentioned that IL1B can inhibit MSC chondrogenesis.

1000pg/mL of IL-1B resulted in catabolic effects.  Doses between 50-1000 were generally not tested. In some metrics catabolic effects were noted at 100pg/mL. There was a slight increase in Type II collagen at 50pg/mL(with a large inhibition at 1000pg/mL illustrating the importance of inhibiting IL-1B for height growth if levels are too high).  So slight IL1B may still be beneficial for mesenchymal chondrogenesis.

"MSC were also induced to differentiate in pellet in the absence or presence of IL-1β during the entire culture time. Histological analyses indicated that tissues formed by BM-MSC exposed to low doses of IL-1β (10 and 50 pg/mL) were more intensely stained for cartilage specific matrix than tissues formed in absence of IL-1β, while those exposed to high doses (≥ 250 pg/mL) were less intensely stained"

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