Ulnar-Sided Wrist Pain in the Athlete: Sport-Specific Demands, Clinical Presentation, and Management Options.

ABSTRACT: Ulnar-sided wrist injuries are common in sports that require repeated pronosupination, wrist radial/ulnar deviation, axial loading, and gripping equipment. Common anatomic structures affected include the triangular fibrocartilage complex, extensor carpi ulnaris tendon, distal radioulnar and ulnocarpal joints, and hamate bone. Presenting symptoms include pain with activity, swelling, possible snapping or clicking, and reproduction of symptoms with provocative maneuvers. Imaging may confirm or rule out pathologies, but abnormal findings also may present in asymptomatic athletes. Initial treatment is usually nonoperative with splinting, load management, activity modification, strengthening the components of the kinetic chain of the particular sport, and pain management. Surgery is usually indicated in ulnar-wrist pain pathology such as hook of hamate fractures and required in associated instability. Future research should address specific treatment and rehabilitation protocols, emphasizing the complete kinetic chain along with the injured wrist.

Human iPSCs can be differentiated into notochordal cells that reduce intervertebral disc degeneration in a porcine model.

Introduction: As many as 80% of the adult population experience back pain at some point in their lifetimes. Previous studies have indicated a link between back pain and intervertebral disc (IVD) degeneration. Despite decades of research, there is an urgent need for robust stem cell therapy targeting underlying causes rather than symptoms. It has been proposed that notochordal cells (NCs) appear to be the ideal cell type to regenerate the IVD: these cells disappear in humans as they mature, are replaced by nucleus pulposus (NP) cells, and their disappearance correlates with the initiation of degeneration of the disc. Human NCs are in short supply, thus here aimed for generation of notochordal-like cells from induced pluripotent cells (iPSCs). Methods: Human iPSCs were generated from normal dermal fibroblasts by transfecting plasmids encoding for six factors: OCT4, SOX2, KLF4, L-MYC, LIN28, and p53 shRNA. Then the iPSCs were treated with GSK3i to induce differentiation towards Primitive Streak Mesoderm (PSM). The differentiation was confirmed by qRT-PCR and immunofluorescence. PSM cells were transfected with Brachyury (Br)-encoding plasmid and the cells were encapsulated in Tetronic-tetraacrylate-fibrinogen (TF) hydrogel that mimics the NP environment (G'=1kPa), cultured in hypoxic conditions (2% O2) and with specifically defined growth media. The cells were also tested in vivo in a large animal model. IVD degeneration was induced after an annular puncture in pigs, 4 weeks later the cells were injected and IVDs were analyzed at 12 weeks after the injury using MRI, gene expression analysis and histology. Results: After short-term exposure of iPSCs to GSK3i there was a significant change in cell morphology, Primitive Streak Mesoderm (PSM) markers (Brachyury, MIXL1, FOXF1) were upregulated and markers of pluripotency (Nanog, Oct4, Sox2) were downregulated, both compared to the control group. PSM cells nucleofected with Br (PSM-Br) cultured in TF hydrogels retained the NC phenotype consistently for up to 8 weeks, as seen in the gene expression analysis. PSM-Br cells were co-cultured with bone marrow (BM)-derived mesenchymal stem cells (MSCs) which, with time, expressed the NC markers in higher levels, however the levels of expression in BM-MSCs alone did not change. Higher expression of NC and NP marker genes in human BM-MSCs was found to be induced by iNC-condition media (iNC-CM) than porcine NC-CM. The annular puncture induced IVD degeneration as early as 2 weeks after the procedure. The injected iNCs were detected in the degenerated discs after 8 weeks in vivo. The iNC-treated discs were found protected from degeneration. This was evident in histological analysis and changes in the pH levels, indicative of degeneration state of the discs, observed using qCEST MRI. Immunofluorescence stains show that their phenotype was consistent with the in vitro study, namely they still expressed the notochordal markers Keratin 18, Keratin 19, Noto and Brachyury. Conclusion: In the present study, we report a stepwise differentiation method to generate notochordal cells from human iPSCs. These cells not only demonstrate a sustainable notochordal cell phenotype in vitro and in vivo, but also show the functionality of notochordal cells and have protective effect in case of induced disc degeneration and prevent the change in the pH level of the injected IVDs. The mechanism of this effect could be suggested via the paracrine effect on resident cells, as it was shown in the in vitro studies with MSCs.

Bone-chip system to monitor osteogenic differentiation using optical imaging.

Human organoids and organ-on-chip systems to predict human responses to new therapies and for the understanding of disease mechanisms are being more commonly used in translational research. We have developed a bone-chip system to study osteogenic differentiation in vitro, coupled with optical imaging approach which provides the opportunity of monitoring cell survival, proliferation and differentiation in vitro without the need to terminate the culture. We used the mesenchymal stem cell (MSC) line over-expressing bone morphogenetic protein-2 (BMP-2), under Tet-Off system, and luciferase reporter gene under constitutive promoter. Cells were seeded on chips and supplemented with osteogenic medium. Flow of media was started 24 h later, while static cultures were performed using media reservoirs. Cells grown on the bone-chips under constant flow of media showed enhanced survival/proliferation, comparing to the cells grown in static conditions; luciferase reporter gene expression and activity, reflecting the cell survival and proliferation, was quantified using bioluminescence imaging and a significant advantage to the flow system was observed. In addition, the flow had positive effect on osteogenic differentiation, when compared with static cultures. Quantitative fluorescent imaging, performed using the osteogenic extra-cellular matrix-targeted probes, showed higher osteogenic differentiation of the cells under the flow conditions. Gene expression analysis of osteogenic markers confirmed the osteogenic differentiation of the MSC-BMP2 cells. Immunofluorescent staining performed against the Osteocalcin, Col1, and BSP markers illustrated robust osteogenic differentiation in the flow culture and lessened differentiation in the static culture. To sum, the bone-chip allows monitoring cell survival, proliferation, and osteogenic differentiation using optical imaging.

Systemic administration of mesenchymal stem cells combined with parathyroid hormone therapy synergistically regenerates multiple rib fractures.

BACKGROUND: A devastating condition that leads to trauma-related morbidity, multiple rib fractures, remain a serious unmet clinical need. Systemic administration of mesenchymal stem cells (MSCs) has been shown to regenerate various tissues. We hypothesized that parathyroid hormone (PTH) therapy would enhance MSC homing and differentiation, ultimately leading to bone formation that would bridge rib fractures. METHODS: The combination of human MSCs (hMSCs) and a clinically relevant PTH dose was studied using immunosuppressed rats. Segmental defects were created in animals' fifth and sixth ribs. The rats were divided into four groups: a negative control group, in which animals received vehicle alone; the PTH-only group, in which animals received daily subcutaneous injections of 4 µg/kg teriparatide, a pharmaceutical derivative of PTH; the hMSC-only group, in which each animal received five injections of 2 × 106 hMSCs; and the hMSC + PTH group, in which animals received both treatments. Longitudinal in vivo monitoring of bone formation was performed biweekly using micro-computed tomography (µCT), followed by histological analysis. RESULTS: Fluorescently-dyed hMSCs were counted using confocal microscopy imaging of histological samples harvested 8 weeks after surgery. PTH significantly augmented the number of hMSCs that homed to the fracture site. Immunofluorescence of osteogenic markers, osteocalcin and bone sialoprotein, showed that PTH induced cell differentiation in both exogenously administered cells and resident cells. µCT scans revealed a significant increase in bone volume only in the hMSC + PTH group, beginning by the 4th week after surgery. Eight weeks after surgery, 35% of ribs in the hMSC + PTH group had complete bone bridging, whereas there was complete bridging in only 6.25% of ribs (one rib) in the PTH-only group and in none of the ribs in the other groups. Based on the µCT scans, biomechanical analysis using the micro-finite element method demonstrated that the healed ribs were stiffer than intact ribs in torsion, compression, and bending simulations, as expected when examining bone callus composed of woven bone. CONCLUSIONS: Administration of both hMSCs and PTH worked synergistically in rib fracture healing, suggesting this approach may pave the way to treat multiple rib fractures as well as additional fractures in various anatomical sites.

Quantitative chemical exchange saturation transfer MRI of intervertebral disc in a porcine model.

PURPOSE: Previous studies have associated low pH in intervertebral discs (IVDs) with discogenic back pain. The purpose of this study was to determine whether quantitative CEST (qCEST) MRI can be used to detect pH changes in IVDs in vivo. METHODS: The exchange rate ksw between glycosaminoglycan (GAG) protons and water protons was determined from qCEST analysis. Its dependence on pH value was investigated in GAG phantoms with varying pH and concentrations. The relationship between ksw and pH was studied further in vivo in a porcine model on a 3T MR scanner and validated using a pH meter. Sodium lactate was injected into the IVDs to induce various pH values within the discs ranging from 5 to 7. RESULTS: Phantom and animal results revealed that ksw measured using qCEST MRI is highly correlated with pH level. In the animal studies, the relationship can be described as ksw =9.2 × 106 × 10-pH + 196.9, R2 = 0.7883. CONCLUSION: The exchange rate between GAG and water protons determined from qCEST MRI is closely correlated with pH value. This technique has the potential to noninvasively measure pH in the IVDs of patients with discogenic pain. Magn Reson Med 76:1677-1683, 2016. © 2016 International Society for Magnetic Resonance in Medicine.

Human Induced Pluripotent Stem Cells Differentiate Into Functional Mesenchymal Stem Cells and Repair Bone Defects.

: Mesenchymal stem cells (MSCs) are currently the most established cells for skeletal tissue engineering and regeneration; however, their availability and capability of self-renewal are limited. Recent discoveries of somatic cell reprogramming may be used to overcome these challenges. We hypothesized that induced pluripotent stem cells (iPSCs) that were differentiated into MSCs could be used for bone regeneration. Short-term exposure of embryoid bodies to transforming growth factor-ß was used to direct iPSCs toward MSC differentiation. During this process, two types of iPSC-derived MSCs (iMSCs) were identified: early (aiMSCs) and late (tiMSCs) outgrowing cells. The transition of iPSCs toward MSCs was documented using MSC marker flow cytometry. Both types of iMSCs differentiated in vitro in response to osteogenic or adipogenic supplements. The results of quantitative assays showed that both cell types retained their multidifferentiation potential, although aiMSCs demonstrated higher osteogenic potential than tiMSCs and bone marrow-derived MSCs (BM-MSCs). Ectopic injections of BMP6-overexpressing tiMSCs produced no or limited bone formation, whereas similar injections of BMP6-overexpressing aiMSCs resulted in substantial bone formation. Upon orthotopic injection into radial defects, all three cell types regenerated bone and contributed to defect repair. In conclusion, MSCs can be derived from iPSCs and exhibit self-renewal without tumorigenic ability. Compared with BM-MSCs, aiMSCs acquire more of a stem cell phenotype, whereas tiMSCs acquire more of a differentiated osteoblast phenotype, which aids bone regeneration but does not allow the cells to induce ectopic bone formation (even when triggered by bone morphogenetic proteins), unless in an orthotopic site of bone fracture. SIGNIFICANCE: Mesenchymal stem cells (MSCs) are currently the most established cells for skeletal tissue engineering and regeneration of various skeletal conditions; however, availability of autologous MSCs is very limited. This study demonstrates a new method to differentiate human fibroblast-derived induced pluripotent stem cells (iPSCs) to cells with MSC properties, which we comprehensively characterized including differentiation potential and transcriptomic analysis. We showed that these iPS-derived MSCs are able to regenerate nonunion bone defects in mice more efficiently than bone marrow-derived human MSCs when overexpressing BMP6 using a nonviral transfection method.