Chondrogenic potential of human synovial mesenchymal stem cells in alginate

OBJECTIVE: In a recent study, we demonstrated that mesenchymal stem cells (MSCs) derived from the synovial membranes of bovine shoulder joints could differentiate into chondrocytes when cultured in alginate. The purpose of the present study was to establish the conditions under which synovial MSCs derived from aging human donors can be induced to undergo chondrogenic differentiation using the same alginate system. METHODS: MSCs were obtained by digesting the knee-joint synovial membranes of osteoarthritic human donors (aged 59-76 years), and expanded in monolayer cultures. The cells were then seeded at a numerical density of 4x10(6)/ml within discs of 2% alginate, which were cultured in serum-containing or serum-free medium (the latter being supplemented with 1% insulin, transferrin, selenium (ITS). The chondrogenic differentiation capacity of the cells was tested by exposing them to the morphogens transforming growth factor-beta1 (TGF-beta1), TGF-beta2, TGF-beta3, insulin-like growth factor-1 (IGF-1), bone morphogenetic protein-2 (BMP-2) and BMP-7, as well as to the synthetic glucocorticoid dexamethasone. The relative mRNA levels of collagen types I and II, of aggrecan and of Sox9 were determined quantitatively by the real-time polymerase chain reaction (PCR). The extracellular deposition of proteoglycans was evaluated histologically after staining with Toluidine Blue, and that of type-II collagen by immunohistochemistry. RESULTS: BMP-2 induced the chondrogenic differentiation of human synovial MSCs in a dose-dependent manner. The response elicited by BMP-7 was comparable. Both of these agents were more potent than TGF-beta1. A higher level of BMP-2-induced chondrogenic differentiation was achieved in the absence than in the presence of serum. In the presence of dexamethasone, the BMP-2-induced expression of mRNAs for aggrecan and type-II collagen was suppressed; the weaker TGF-beta1-induced expression of these chondrogenic markers was not obviously affected. CONCLUSIONS: We have demonstrated that synovial MSCs derived from the knee joints of aging human donors possess chondrogenic potential. Under serum-free culturing conditions and in the absence of dexamethasone, BMP-2 and BMP-7 were the most potent inducers of this transformation process.

Site-1 protease is essential for endochondral bone formation in mice

Site-1 protease (S1P) has an essential function in the conversion of latent, membrane-bound transcription factors to their free, active form. In mammals, abundant expression of S1P in chondrocytes suggests an involvement in chondrocyte function. To determine the requirement of S1P in cartilage and bone development, we have created cartilage-specific S1P knockout mice (S1P(cko)). S1P(cko) mice exhibit chondrodysplasia and a complete lack of endochondral ossification even though Runx2 expression, Indian hedgehog signaling, and osteoblastogenesis is intact. However, there is a substantial increase in chondrocyte apoptosis in the cartilage of S1P(cko) mice. Extraction of type II collagen is substantially lower from S1P(cko) cartilage. In S1P(cko) mice, the collagen network is disorganized and collagen becomes entrapped in chondrocytes. Ultrastructural analysis reveals that the endoplasmic reticulum (ER) in S1P(cko) chondrocytes is engorged and fragmented in a manner characteristic of severe ER stress. These data suggest that S1P activity is necessary for a specialized ER stress response required by chondrocytes for the genesis of normal cartilage and thus endochondral ossification.

In vitro resistance of articular chondrocytes to 5-Aminolevulinic acid based photodynamic therapy

OBJECTIVE: 5-Aminolevulinic acid based photodynamic therapy (5-ALA-PDT) has revealed promising results in the treatment of inflammatory joint diseases due to the sensitivity of inflamed synovial tissue. For 5-ALA-PDT to be safe and beneficial for intra-articular applications, resistance of chondrocytes is essential to prevent cartilage damage. As no data yet exist, the aim of the present study was to assess in vitro the response of the chondrocytes to 5-ALA-PDT and to compare with osteoblasts and synovial tissue derived cells. METHODS: Bovine articular chondrocytes, osteoblasts, and synovial cells were subjected to 5-ALA-PDT in cell culture. The PpIX accumulation and the function of the cells were assessed for up to 12 days. RESULTS: Bovine chondrocytes showed lower PpIX fluorescence upon incubation with 5-ALA (0.0-2.0 mM) for 4 hours as compared to osteoblasts and synovial cells suggesting a low PpIX accumulation. After incubation with 0.5 mM 5-ALA and application of light at a dose of 20 J/cm2, chondrocytes were functionally not affected (collagen type II and aggrecan mRNA, glycosaminoglycan synthesis) whereas a decrease in the proportion of viable cells was observed in osteoblasts and synovial cells (2+/-2% and 14+/-8%, respectively; chondrocytes 91+/-13%). Chondrocytes showed a 58% reduction of 5-ALA uptake using [3H]5-ALA as compared to osteoblasts and a lower mitochondrial content as assessed by the activity of the mitochondrial marker enzyme citrate synthase (9.2+/- 3.6 mU/mg protein) than osteoblasts (32.6+/-10.5 mU/mg) and synovial cells (60.0+/-10.8 mU/mg). The reduced uptake of 5-ALA and/or the low mitochondrial content, an adaptation to their in vivo environment and the site of PpIX synthesis, presumably explains the lower PpIX content in chondrocytes and their resistance against 5-ALA-PDT. CONCLUSION: 5-ALA-PDT might represent a treatment strategy in inflammatory joint diseases without endangering the cartilage function. However, further in vitro and in vivo experiments are required to confirm this data in the authentic environment of chondrocytes, the articular cartilage.

Hypoxic expansion promotes the chondrogenic potential of articular chondrocytes

For cell-based cartilage repair strategies, an ex vivo expansion phase is required to obtain sufficient numbers of cells needed for therapy. Although recent reports demonstrated the central role of oxygen for the function and differentiation of chondrocytes, a beneficial effect of low oxygen concentrations during the expansion of the cells to further improve their chondrogenic capacity has not been investigated.Therefore, freshly harvested bovine articular chondrocytes were grown in two-dimensional monolayer cultures at 1.5% and 21% O2 and redifferentiation was subsequently induced in three-dimensional micromass cultures at 1.5%, 5%, and 21% O2. Cells expanded at 1.5% O2 were characterized by low citrate synthase (aerobic energy metabolism)--and high LDH (anaerobic energy metabolism-activities,suggesting an anaerobic energy metabolism. Collagen type II mRNA was twofold higher in cells expanded at 1.5% as compared to expansion at 21% O2. Micromass cultures grown at 21% O2 showed up to a twofold increase in the tissue content of glycosaminoglycans when formed with cells expanded at 1.5% instead of 21% O2. However, no differences in the levels of transcripts and in the staining for collagen type II protein were observed in these micromass cultures. Hypoxia (1.5% and 5% O2) applied during micromass cultures gave rise to tissues with low contents of glycosaminoglycans only. In vivo, the chondrocytes are adapted to a hypoxic environment. Taking this into account, by applying 1.5% O2 in the expansion phase in the course of cell-based cartilage repair strategies, may result in a repair tissue with higher quality by increasing the content of glycosaminoglycans.

Hypovolemia contributes to the pathogenesis of orthostatic hypotension in patients with diabetes mellitus

PURPOSE: To investigate whether body sodium content and blood volume contribute to the pathogenesis of orthostatic hypotension in patients with diabetes mellitus. SUBJECTS AND METHODS: Exchangeable sodium, plasma and blood volumes, and catecholamine, renin, and aldosterone levels were assessed in 10 patients with Type II diabetes mellitus who had orthostatic hypotension and control groups of 40 diabetic patients without orthostatic hypotension and 40 normal subjects of similar age and sex. In subgroups, clinical tests of autonomic function and cardiovascular reactivity to norepinephrine and angiotensin II infusions were performed. RESULTS: In diabetic patients with orthostatic hypotension, mean (+/- SD) supine blood pressure was 165/98 +/- 27/12 mm Hg (P <0.05 compared with other groups) and mean upright blood pressure was 90/60 +/- 38/18 mm Hg. Compared with controls, diabetic patients with orthostatic hypotension had a 10% lower blood volume. They also had less exchangeable sodium than patients with diabetes who did not have orthostatic hypotension (P <0.01). Compared with both groups of controls, diabetic patients with orthostatic hypotension had decreased 24-hour urinary norepinephrine excretion and a reduced diastolic blood pressure response to handgrip (P <0.05). Moreover, they displayed reduced products of exchangeable sodium or blood volume and sympathetic function indexes. Cardiovascular pressor reactivity to norepinephrine was enhanced (P <0.01) and beat-to-beat variation decreased (P <0.01) in both groups of diabetic patients. Microvascular complications were more prevalent in the diabetic patients with orthostatic hypotension (90% vs 35%). CONCLUSIONS: Patients who have Type II diabetes mellitus and orthostatic hypotension are hypovolemic and have sympathoadrenal insufficiency; both factors contribute to the pathogenesis of orthostatic hypotension.

Three-dimensional consensus structure of sst2-selective somatostatin (SRIF) antagonists by NMR

The three-dimensional NMR structures of seven octapeptide analogs of somatostatin (SRIF), based on octreotide, with the basic sequence H-Cpa/Phe2-c[DCys3-Xxx7-DTrp/DAph(Cbm)8-Lys9-Thr10-Cys14]-Yyy-NH2 (the numbering refers to the position in native SRIF), with Xxx7 being Aph(Cbm)/Tyr/Agl(NMe,benzoyl) and Yyy being Nal/DTyr/Thr, are presented here. Most of these analogs exhibit potent and highly selective binding to sst2 receptors, and all of the analogs are antagonists inhibiting receptor signaling. Based on their consensus 3D structure, the pharmacophore of the sst2-selective antagonist has been defined. The pharmacophore involves the side chains of Cpa2, DTrp/DAph(Cbm)8, and Lys9, with the backbone for most of the sst2-selective antagonists comprised a Type-II' beta-turn. Hence, the sst2-selective antagonist pharmacophore is very similar to the sst2-selective agonist pharmacophore previously described.