Metabolomics and cytokine profiling of mesenchymal stromal cells identify markers predictive of T-cell suppression.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) have shown great promise in the field of regenerative medicine, as many studies have shown that MSCs possess immunomodulatory function. Despite this promise, no MSC therapies have been licensed by the Food and Drug Administration. This lack of successful clinical translation is due in part to MSC heterogeneity and a lack of critical quality attributes. Although MSC indoleamine 2,3-dioxygnease (IDO) activity has been shown to correlate with MSC function, multiple predictive markers may be needed to better predict MSC function. METHODS: Three MSC lines (two bone marrow-derived, one induced pluripotent stem cell-derived) were expanded to three passages. At the time of harvest for each passage, cell pellets were collected for nuclear magnetic resonance (NMR) and ultra-performance liquid chromatography mass spectrometry (MS), and media were collected for cytokine profiling. Harvested cells were also cryopreserved for assessing function using T-cell proliferation and IDO activity assays. Linear regression was performed on functional data against NMR, MS and cytokines to reduce the number of important features, and partial least squares regression (PLSR) was used to obtain predictive markers of T-cell suppression based on variable importance in projection scores. RESULTS: Significant functional heterogeneity (in terms of T-cell suppression and IDO activity) was observed between the three MSC lines, as were donor-dependent differences based on passage. Omics characterization revealed distinct differences between cell lines using principal component analysis. Cell lines separated along principal component one based on tissue source (bone marrow-derived versus induced pluripotent stem cell-derived) for NMR, MS and cytokine profiles. PLSR modeling of important features predicted MSC functional capacity with NMR (R2 = 0.86), MS (R2 = 0.83), cytokines (R2 = 0.70) and a combination of all features (R2 = 0.88). CONCLUSIONS: The work described here provides a platform for identifying markers for predicting MSC functional capacity using PLSR modeling that could be used as release criteria and guide future manufacturing strategies for MSCs and other cell therapies.

Morphological landscapes from high content imaging reveal cytokine priming strategies that enhance mesenchymal stromal cell immunosuppression.

Successful clinical translation of mesenchymal stromal cell (MSC) products has not been achieved in the United States and may be in large part due to MSC functional heterogeneity. Efforts have been made to identify "priming" conditions that produce MSCs with consistent immunomodulatory function; however, challenges remain with predicting and understanding how priming impacts MSC behavior. The purpose of this study was to develop a high throughput, image-based approach to assess MSC morphology in response to combinatorial priming treatments and establish morphological profiling as an effective approach to screen the effect of manufacturing changes (i.e., priming) on MSC immunomodulation. We characterized the morphological response of multiple MSC lines/passages to an array of Interferon-gamma (IFN-γ) and tumor necrosis factor-⍺ (TNF-⍺) priming conditions, as well as the effects of priming on MSC modulation of activated T cells and MSC secretome. Although considerable functional heterogeneity, in terms of T-cell suppression, was observed between different MSC lines and at different passages, this heterogeneity was significantly reduced with combined IFN-γ/TNF-⍺ priming. The magnitude of this change correlated strongly with multiple morphological features and was also reflected by MSC secretion of immunomodulatory factors, for example, PGE2, ICAM-1, and CXCL16. Overall, this study further demonstrates the ability of priming to enhance MSC function, as well as the ability of morphology to better understand MSC heterogeneity and predict changes in function due to manufacturing.

Biomechanical consequences of adding plantar fascia release to metatarsal osteotomies: Changes in forefoot plantar pressures.

Destruction of the normal metatarsal arch by a long metatarsal is often a cause for metatarsalgia. When surgery is warranted, distal oblique, or proximal dorsiflexion osteotomies of the long metatarsal bones are commonly used. The plantar fascia has anatomical connection to all metatarsal heads. There is controversial scientific evidence on the effect of plantar fascia release on forefoot biomechanics. In this cadaveric biomechanical study, we hypothesized that plantar fascia release would augment the plantar metatarsal pressure decreasing effects of two common second metatarsal osteotomy techniques. Six matched pairs of foot and ankle specimens were mounted on a pressure mat loading platform. Two randomly assigned surgery groups, which had received either distal oblique, or proximal dorsiflexion osteotomy of the second metatarsal, were evaluated before and after plantar fasciectomy. Specimens were loaded up to a ground reaction force of 400 N at varying Achilles tendon forces. Average pressures, peak pressures, and contact areas were analyzed. Supporting our hypothesis, average pressures under the second metatarsal during 600 N Achilles load were decreased by plantar fascia release following proximal osteotomy (p < 0.05). However contrary to our hypothesis, peak pressures under the second metatarsal were significantly increased by plantar fascia release following modified distal osteotomy, under multiple Achilles loading conditions (p < 0.05). Plantar fasciotomy should not be added to distal metatarsal osteotomy in the treatment of metatarsalgia. If proximal dorsiflexion osteotomy would be preferred, plantar fasciotomy should be approached cautiously not to disturb the forefoot biomechanics. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:800-804, 2017.

Biomechanical benefits of anterior offsetting of humeral head component in posteriorly unstable total shoulder arthroplasty: A cadaveric study.

Restoration of joint stability during total shoulder arthroplasty can be challenging in the face of severe glenoid retroversion. A novel technique of humeral head component anterior-offsetting has been proposed to address posterior instability. We evaluated the biomechanical benefits of this technique in cadaveric specimens. Total shoulder arthroplasty was performed in 14 cadaveric shoulders from 7 donors. Complementary shoulders were assigned to either 10° or 20° glenoid retroversion, with retroversion created by eccentric reaming. Two humeral head component offset positions were tested in each specimen: The anatomic (posterior) and anterior (reverse). With loads applied to the rotator cuff and deltoid, joint contact pressures and the force and energy required for posterior humeral head translation were measured. The force and energy required to displace the humeral head posteriorly increased significantly with the anterior offset position compared to the anatomic offset position. The joint contact pressures were significantly shifted anteriorly, and the joint contact area significantly increased with the anterior offset position. Anterior offsetting of the humeral head component increased the resistance to posterior humeral head translation, shifted joint contact pressures anteriorly, and increased joint contact area, thus, potentially increasing the joint stability in total shoulder arthroplasty with simulated glenoid retroversion.

Comparison of Proximal and Distal Oblique Second Metatarsal Osteotomies with Varying Achilles Tendon Tension: Biomechanical Study in a Cadaver Model.

BACKGROUND: The optimal surgery for reducing pressure under the second metatarsal head to treat metatarsalgia is unknown. We tested our hypothesis that a proximal oblique dorsiflexion osteotomy of the second metatarsal would decrease second-metatarsal plantar pressures in a cadaver model with varying Achilles tendon tension. We also tested the plantar pressure effects of two popular techniques of distal oblique osteotomy. METHODS: Twelve fresh-frozen feet from six cadavers were randomly assigned to either the distal osteotomy group (a classic distal oblique osteotomy followed by a modified distal oblique osteotomy) or proximal metatarsal osteotomy group. Each specimen was tested intact and then after the osteotomy or osteotomies. The feet were loaded with 0, 300, and 600 N of Achilles tendon tension and a 400-N ground reaction force. Plantar pressures were measured by a pressure sensitive mat and analyzed in sections located under each metatarsal. RESULTS: The proximal metatarsal osteotomy significantly reduced average pressures beneath the second metatarsal head during both 300 and 600 N of Achilles tendon loading by an average of 19.4 and 29.7 kPa, respectively (p < 0.05). The modified distal oblique osteotomy significantly decreased these pressures during 600 N of Achilles tendon loading, by a mean of 20.2 kPa, which was to a lesser extent than the proximal metatarsal osteotomy. Interestingly, the classic distal oblique osteotomy was not found to have significant effects on pressures beneath the second metatarsal head. CONCLUSIONS: The proximal oblique dorsiflexion metatarsal osteotomy may be the most effective procedure for decreasing plantar pressures under the second metatarsal. The modified distal oblique osteotomy may be the second most effective. CLINICAL RELEVANCE: The findings of this biomechanical study help shed light on which of the common second metatarsal osteotomies are best for decreasing plantar pressures.