Heparin concentration is critical for cell culture with human platelet lysate.

BACKGROUND AIMS: Culture media for mesenchymal stromal cells (MSCs) are generally supplemented with fetal bovine serum. Human platelet lysate (hPL) has been proven to be a very effective alternative without the risk of xenogeneic infections or immune reactions. In contrast to fetal bovine serum, hPL comprises plasma, and anticoagulants-usually unfractionated heparin (UFH)-need to be added to prevent gel formation. METHODS: Cultures of MSCs in hPL media with various concentrations of UFH and enoxaparin, a low-molecular-weight heparin (LMWH), were systematically compared with regard to proliferation, fibroblastoid colony-forming unit frequency, immunophenotype and in vitro differentiation. RESULTS: At least 0.61 IU/mL UFH or 0.024 mg/mL LMWH was necessary for reliable prevention of coagulation of hPL pools used in this study. Higher concentrations impaired cellular proliferation in a dose-dependent manner even without benzyl alcohol, which is commonly added to heparins as a bacteriostatic agent. Colony-forming unit frequency was also reduced at higher heparin concentrations, particularly with LMWH, whereas no significant effect was observed on cellular morphology or immunophenotype. High concentrations of heparins reduced the in vitro differentiation toward adipogenic and osteogenic lineages. CONCLUSIONS: Heparin concentration is critical for culture of MSCs in hPL media; this is of particular relevance for cellular therapy where cell culture procedures need to be optimized and standardized.

Ultrafast high-harmonic nanoscopy of magnetization dynamics.

Light-induced magnetization changes, such as all-optical switching, skyrmion nucleation, and intersite spin transfer, unfold on temporal and spatial scales down to femtoseconds and nanometers, respectively. Pump-probe spectroscopy and diffraction studies indicate that spatio-temporal dynamics may drastically affect the non-equilibrium magnetic evolution. Yet, direct real-space magnetic imaging on the relevant timescales has remained challenging. Here, we demonstrate ultrafast high-harmonic nanoscopy employing circularly polarized high-harmonic radiation for real-space imaging of femtosecond magnetization dynamics. We map quenched magnetic domains and localized spin structures in Co/Pd multilayers with a sub-wavelength spatial resolution down to 16 nm, and strobosocopically trace the local magnetization dynamics with 40 fs temporal resolution. Our compact experimental setup demonstrates the highest spatio-temporal resolution of magneto-optical imaging to date. Facilitating ultrafast imaging with high sensitivity to chiral and linear dichroism, we envisage a wide range of applications spanning magnetism, phase transitions, and carrier dynamics.

Standardised method of surgical treatment of chronic leg ulcers.

Healing and recurrence rates were compared in leg ulcers of different aetiology in 385 patients with 406 chronic leg ulcers. Standard treatment was excision of the ulcer followed by meshed split-skin grafting and correction of superficial venous insufficiency in the area. The median age of the patients was 75 years (range 16-95). After one year 345 patients with 357 leg ulcers were alive. Overall healing rate was 64% (227 in 357 legs) after one year. The best results were achieved in traumatic ulcers (31 in 36 ulcers) and worst for the arterial ulcers (4 in 20 ulcers). Recurrence rate in venous and venous/ischaemic ulcers was 14% (33 in 235 legs) and 8% (n=3) in the traumatic ulcers. Vasculitic ulcers tend to recur 59% (n=10), but the graft does relieve pain.

Donor age of human platelet lysate affects proliferation and differentiation of mesenchymal stem cells.

The regenerative potential declines upon aging. This might be due to cell-intrinsic changes in stem and progenitor cells or to influences by the microenvironment. Mesenchymal stem cells (MSC) raise high hopes in regenerative medicine. They are usually culture expanded in media with fetal calf serum (FCS) or other serum supplements such as human platelet lysate (HPL). In this study, we have analyzed the impact of HPL-donor age on culture expansion. 31 single donor derived HPLs (25 to 57 years old) were simultaneously compared for culture of MSC. Proliferation of MSC did not reveal a clear association with platelet counts of HPL donors or growth factors concentrations (PDGF-AB, TGF-ß1, bFGF, or IGF-1), but it was significantly higher with HPLs from younger donors (<35 years) as compared to older donors (>45 years). Furthermore, HPLs from older donors increased activity of senescence-associated beta-galactosidase (SA-ßgal). HPL-donor age did not affect the fibroblastoid colony-forming unit (CFU-f) frequency, immunophenotype or induction of adipogenic differentiation, whereas osteogenic differentiation was significantly lower with HPLs from older donors. Concentrations of various growth factors (PDGF-AB, TGF-ß1, bFGF, IGF-1) or hormones (estradiol, parathormone, leptin, 1,25 vitamin D3) were not associated with HPL-donor age or MSC growth. Taken together, our data support the notion that aging is associated with systemic feedback mechanisms acting on stem and progenitor cells, and this is also relevant for serum supplements in cell culture: HPLs derived from younger donors facilitate enhanced expansion and more pronounced osteogenic differentiation.

Achilles tendon lengthening, the panacea for plantar forefoot ulceration?

BACKGROUND: Recent reports on Achilles tendon lengthening (ATL) have documented rapid healing of chronic plantar neuropathic forefoot ulcers. METHODS: Sixty-eight patients with 75 ulcerated neuropathic feet (63 patients with diabetes with 69 ulcerated feet) underwent ATL and were retrospectively studied for chronic plantar ulceration in the forefoot and reduced dorsiflexion range of motion at the ankle. Median duration of ulcers was 48 months (range 11-84 months) in 16 forefoot amputation stumps and 11 months (range 3-84 months) for the rest of the patients. RESULTS: Healing of the ulcers was obtained in 68/75 feet (91%). At a median follow-up of 12 months (3-26 months), 4 had never healed, 5/10 recurrent ulcers and 6/11 transfer ulcers (localized to the heel) had not healed, that is, a healing rate of 60/75 (80%). Acute transfer ulcers to the heel occurred in 47% of patients with complete anaesthesia of the heel pad. Late heel ulceration occurred in 14% of those with extreme ankle dorsiflexion (>15 degrees). Failure to heal or ulcer recurrence occurred more frequently at dorsiflexion <10 degrees (33%). Rupture of the Achilles tendon occurred in 7 feet (10%). Charcot event was experienced in 3 (4%). CONCLUSION: Lengthening of the Achilles tendon is effective in healing plantar neuropathic ulceration. Patients with complete anaesthesia of the heel pad should not undergo this procedure and extreme dorsiflexion should be avoided because of the risk of heel ulceration. Moreover, the procedure should be performed only in a multidisciplinary setting for immediate dealing with complications and for surveillance and treatment of future ulceration.