Potential of Continuous Local Antibiotic Perfusion Therapy for Fracture-Related Infections: A Case Series

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Additional file 1 of DAIR in treating chronic PJI after total knee arthroplasty using continuous local antibiotic perfusion therapy: a case series study

Supplementary Material 1: Fig. A Dual-lumen tubes (Salam samp tube®: Nihon Covidien Co.

Nano-fiber plugs induce osteogenesis of human MSCs.

<p>Healthy donor-derived MSCs were seeded onto plastic plates (A-C) or injected into the nano-fiber plugs (D-I), and then cultured in MSCGM for 1 day (D), 7 days (E, G), 14 days (F, H) and 28 days (A-C, I). Then, the expression levels of RUNX2 (A, D, G), osteocalcin (B, E, H), and DMP-1 (C, F, I) were evaluated immunohistochemically. Representative results of three experiments are shown. (J) Healthy donor-derived MSCs were cultured in MSCGM onto plastic plates (two-dimensional) or nano-fiber plugs (three-dimensional) for 28 days. After RNA extraction, <i>MEPE</i> expression in the 4 groups of healthy donor-derived MSCswas evaluated by real-time PCR. *p<0.05 vs. two-dimensional culture by paired <i>t</i>-test. Scale bars, 50 μm.</p

The hierarchy of gene expression in MSCs during differentiaion into osteoblasts, chondrocytes, and osteocytes.

<p>MSCs differentiate into osteoblasts or chondrocytes under the regulation of their master regulators, RUNX2 or SOX9, respectively. A part of chondrocytes also differentiate into osteoblasts.</p

Effects of lactic acid on cell functions of human MSCs.

Healthy donor-derived MSCs seeded onto plastic plates were cultured in MSCGM with lactic acid. (A) Proliferative capacity assessed by measurement of 3H thymidine uptake after 1 day (n = 3). (B) Propidium iodide-positive apoptotic cells and WST-8-positive viable cells on Day 7 (n = 5). (C) ALP activity on Day 7 (n = 3), and (D) production of sGAG on Day 28 (n = 3). Data are mean±SD. *p<0.05, **p<0.01 vs. 0 mM, by ANOVA with post-hoc Dunnett’s test. n.s.: not significant.</p

Nano-fiber plugs induce chondrogenesis of human MSCs.

(A) Healthy donor-derived MSCs were injected into the center of nano-fiber plugs and cultured in MSCGM for the specified period. The samples were assessed immunohistochemically for the expression of type-II collagen on Days 7 and 28. Representative results of three experiments are shown. Scale bars, 50 μm. (B and C) Human MSCs were cultured in MSCGM onto plastic plates (two-dimensional) or nano-fiber plugs (three-dimensional) for the specified period. After RNA extraction, the expression levels of SOX9 at day 7 (B) and COL10A1 at day 28 (C) in the 4 groups of healthy donor-derived MSCswere assessed by real-time PCR. *pt-test.</p

MSCs cultured on nano-fiber plugs produce minerals and proteoglycan upon differentiation stimulation.

<p>Healthy MSCs were seeded onto plastic plates (A, D) or injected into the center of the nano-fiber plugs (B, C, E, F), and then cultured in OIM (A-C) or CM (D-F) for 28 days. The samples were then fixed with formalin, and then stained with von Kossa stain (A, B, E) or Safranin O (C, D, F). Representative results of three experiments are shown. Scale bars, 50 μm.</p

Nano-fiber plugs induce the differentiation of MSCs derived from patients with RA or OA.

<p>MSCs derived from patients with RA (n = 3) or OA (n = 3) were cultured in MSCGM onto plastic plates (two-dimensional) or nano-fiber plugs (three-dimensional) for 7 (A) or 28 days (B). After RNA extraction, real-time PCR was performed to evaluate the gene expression level. Each line represents one patient. MSCs from healthy donors (n = 6), RA (n = 3) or OA (n = 3) patients were cultured in NFs for 7 days (C) or 28 days (D), and then analyzed as for their gene expression by real-time PCR. n.s.: not significant by ANOVA.</p

Nano-fiber plugs induce morphological changes in MSCs.

<p>Healthy donor-derived MSCs were seeded onto nano-fiber plugs, and then cultured in MSCGM. Scanning electron micrographs taken on Days 7 (A) and 28 (B) are shown. All samples were tested three times, and representative results are shown. Scale bars, 50 μm.</p

Characteristics of hMSCs derived from healthy donors or patients with RA/OA.

<p>Expanded bone marrow cells collected from healthy donors (n = 6) or patients with RA (n = 3), OA (n = 3) were analyzed as for their characteristics. Representative histograms of the cell surface markers on bone marrow cells derived from healthy donors (A), patients with RA (B), OA (C) were shown. Red lines show isotype controls. Gene expression of <i>RUNX2</i> (D) and <i>SOX9</i> (E) in hMSCs was analyzed by real-time PCR. n.s.: not significant by ANOVA.</p