IL-6 counteracts the inhibitory effect of IL-4 on osteogenic differentiation of human adipose stem cells.

Fracture repair is characterized by cytokine production and hypoxia. To better predict cytokine modulation of mesenchymal stem cell (MSC)-aided bone healing, we investigated whether interleukin 4 (IL-4), IL-6, and their combination, affect osteogenic differentiation, vascular endothelial growth factor (VEGF) production, and/or mammalian target of rapamycin complex 1 (mTORC1) activation by MSCs under normoxia or hypoxia. Human adipose stem cells (hASCs) were cultured with IL-4, IL-6, or their combination for 3 days under normoxia (20% O 2 ) or hypoxia (1% O 2 ), followed by 11 days without cytokines under normoxia or hypoxia. Hypoxia did not alter IL-4 or IL-6-modulated gene or protein expression by hASCs. IL-4 alone decreased runt-related transcription factor 2 (RUNX2) and collagen type 1 (COL1) gene expression, alkaline phosphatase (ALP) activity, and VEGF protein production by hASCs under normoxia and hypoxia, and decreased mineralization of hASCs under hypoxia. In contrast, IL-6 increased mineralization of hASCs under normoxia, and enhanced RUNX2 gene expression under normoxia and hypoxia. Neither IL-4 nor IL-6 affected phosphorylation of the mTORC1 effector protein P70S6K. IL-4 combined with IL-6 diminished the inhibitory effect of IL-4 on ALP activity, bone nodule formation, and VEGF production, and decreased RUNX2 and COL1 expression, similar to IL-4 alone, under normoxia and hypoxia. In conclusion, IL-4 alone, but not in combination with IL-6, inhibits osteogenic differentiation and angiogenic stimulation potential of hASCs under normoxia and hypoxia, likely through pathways other than mTORC1. These results indicate that cytokines may differentially affect bone healing and regeneration when applied in isolation or in combination.

Biologically Relevant In Vitro 3D-Model to Study Bone Regeneration Potential of Human Adipose Stem Cells.

Standard cell cultures may not predict the proliferation and differentiation potential of human mesenchymal stromal cells (MSCs) after seeding on a scaffold and implanting this construct in a bone defect. We aimed to develop a more biologically relevant in vitro 3D-model for preclinical studies on the bone regeneration potential of MSCs. Human adipose tissue-derived mesenchymal stromal cells (hASCs; five donors) were seeded on biphasic calcium phosphate (BCP) granules and cultured under hypoxia (1% O2) for 14 days with pro-inflammatory TNFα, IL4, IL6, and IL17F (10 mg/mL each) added during the first three days, simulating the early stages of repair (bone construct model). Alternatively, hASCs were cultured on plastic, under 20% O2 and without cytokines for 14 days (standard cell culture). After two days, the bone construct model decreased total DNA (3.9-fold), COL1 (9.8-fold), and RUNX2 expression (19.6-fold) and metabolic activity (4.6-fold), but increased VEGF165 expression (38.6-fold) in hASCs compared to standard cultures. After seven days, the bone construct model decreased RUNX2 expression (64-fold) and metabolic activity (2.3-fold), but increased VEGF165 (54.5-fold) and KI67 expression (5.7-fold) in hASCs compared to standard cultures. The effect of the bone construct model on hASC proliferation and metabolic activity could be largely mimicked by culturing on BCP alone (20% O2, no cytokines). The effect of the bone construct model on VEGF165 expression could be mimicked by culturing hASCs under hypoxia alone (plastic, no cytokines). In conclusion, we developed a new, biologically relevant in vitro 3D-model to study the bone regeneration potential of MSCs. Our model is likely more suitable for the screening of novel factors to enhance bone regeneration than standard cell cultures.

Polymethyl methacrylate does not adversely affect the osteogenic potential of human adipose stem cells or primary osteoblasts.

Custom-made polymethyl methacrylate (PMMA) bone cement is used to treat cranial bone defects but whether it is cytotoxic is still unsure. Possible PMMA-induced adverse effects in vivo affect mesenchymal stem cells and osteoblasts at the implant site. We aimed to investigate whether PMMA affects osteogenic and osteoclast activation potential of human mesenchymal stem cells and/or osteoblasts. Immediately after polymerization, PMMA was added to cultured human adipose stem cells (hASCs) or human osteoblasts (hOBs). Medium lactate dehydrogenase was measured (day 1), metabolic activity, proliferation, osteogenic and osteoclast-activation marker expression (day 1 and 7), and mineralization (day 14). PMMA did not affect lactate dehydrogenase, KI67 gene expression, or metabolic activity in hASCs and hOBs. PMMA transiently decreased DNA content in hOBs only. PMMA increased COL1 gene expression in hASCs, but decreased RUNX2 in hOBs. PMMA did not affect osteocalcin or alkaline phosphatase (ALP) expression, ALP activity, or mineralization. Only in hOBs, PMMA decreased RANKL/OPG ratio. In conclusion, PMMA is not cytotoxic and does not adversely affect the osteogenic potential of hASCs or hOBs. Moreover, PMMA does not enhance production of osteoclast factors by hASCs and hOBs in vitro. Therefore, PMMA bone cement seems highly suitable to treat patients with cranial bone defects.

Cytokines TNF-α, IL-6, IL-17F, and IL-4 Differentially Affect Osteogenic Differentiation of Human Adipose Stem Cells.

During the initial stages of bone repair, proinflammatory cytokines are released within the injury site, quickly followed by a shift to anti-inflammatory cytokines. The effect of pro- and anti-inflammatory cytokines on osteogenic differentiation of mesenchymal stem cells is controversial. Here, we investigated the effect of the proinflammatory cytokines TNF-α, IL-6, IL-8, and IL-17F and the anti-inflammatory cytokine IL-4 on proliferation and osteogenic differentiation of human adipose stem cells (hASCs). hASCs were treated with TNF-α, IL-6, IL-8, IL-17F, or IL-4 (10 ng/mL) for 72 h mimicking bone repair. TNF-α reduced collagen type I gene expression but increased hASC proliferation and ALP activity. IL-6 also strongly enhanced ALP activity (18-fold), as well as bone nodule formation by hASCs. IL-8 did not affect proliferation or osteogenic gene expression but reduced bone nodule formation. IL-17F decreased hASC proliferation but enhanced ALP activity. IL-4 enhanced osteocalcin gene expression and ALP activity but reduced RUNX2 gene expression and bone nodule formation. In conclusion, all cytokines studied have both enhancing and reducing effects on osteogenic differentiation of hASCs, even when applied for 72 h only. Some cytokines, specifically IL-6, may be suitable to induce osteogenic differentiation of mesenchymal stem cells as a strategy for enhancing bone repair.

Osteostatin-coated porous titanium can improve early bone regeneration of cortical bone defects in rats.

A promising bone graft substitute is porous titanium. Porous titanium, produced by selective laser melting (SLM), can be made as a completely open porous and load-bearing scaffold that facilitates bone regeneration through osteoconduction. In this study, the bone regenerative capacity of porous titanium is improved with a coating of osteostatin, an osteoinductive peptide that consists of the 107-111 domain of the parathyroid hormone (PTH)-related protein (PTHrP), and the effects of this osteostatin coating on bone regeneration were evaluated in vitro and in vivo. SLM-produced porous titanium received an alkali-acid-heat treatment and was coated with osteostatin through soaking in a 100 nM solution for 24 h or left uncoated. Osteostatin-coated scaffolds contained ∼0.1 µg peptide/g titanium, and in vitro 81% was released within 24 h. Human periosteum-derived osteoprogenitor cells cultured on osteostatin-coated scaffolds did not induce significant changes in osteogenic (alkaline phosphatase [ALP], collagen type 1 [Col1], osteocalcin [OCN], runt-related transcription factor 2 [Runx2]), or angiogenic (vascular endothelial growth factor [VEGF]) gene expression; however, it resulted in an upregulation of osteoprotegerin (OPG) gene expression after 24 h and a lower receptor activator of nuclear factor kappa-B ligand (RankL):OPG mRNA ratio. In vivo, osteostatin-coated, porous titanium implants increased bone regeneration in critical-sized cortical bone defects (p=0.005). Bone regeneration proceeded until 12 weeks, and femurs grafted with osteostatin-coated implants and uncoated implants recovered, respectively, 66% and 53% of the original femur torque strength (97±31 and 77±53 N·mm, not significant). In conclusion, the osteostatin coating improved bone regeneration of porous titanium. This effect was initiated after a short burst release and might be related to the observed in vitro upregulation of OPG gene expression by osteostatin in osteoprogenitor cells. Long-term beneficial effects of osteostatin-coated, porous titanium implants on bone regeneration or mechanical strength were not established here and may require optimization of the pace and dose of osteostatin release.

Sublingual [corrected] misoprostol as first-line care for incomplete abortion in Burkina Faso.

OBJECTIVE: To explore 400-µg sublingual misoprostol as primary treatment in lower-level facilities with no previous experience providing postabortion care. METHODS: Women presenting with incomplete abortion were offered a single dose of 400-µg sublingual misoprostol. Incomplete abortion was defined as uterine size consistent with fewer than 12 weeks of gestation, open cervical os, and reports of past or present history of vaginal bleeding. Women returned to the clinic 1 week after misoprostol administration for follow-up. At that time, they were discharged if the uterine evacuation was a success or were offered a second follow-up visit or surgical completion if still incomplete. RESULTS: One-hundred women received misoprostol; outcome data were unavailable for 1 woman. Complete uterine evacuation was achieved for 97 (98.0%) women. Satisfaction was high, with nearly all women indicating that they were "satisfied" (n=57 [57.6%]) or "very satisfied" (n=41 [41.4%]) with their experience. Adverse effects were considered "tolerable" by 72 of 97 (74.2%) women. Ninety-seven of 99 (98.0%) participants indicated that they would choose misoprostol for incomplete abortion care in the future and 95 of 97 (97.9%) stated that they would recommend it to a friend. CONCLUSION: Misoprostol is a viable option for treatment of incomplete abortion at mid-level facilities.