Bone marrow-derived mesenchymal stromal cells and platelet-rich plasma on a collagen matrix to improve fascial healing.

OBJECTIVE: To demonstrate improved healing of a midline laparotomy after application of mesenchymal stromal cells and platelet-rich plasma on a collagen matrix and introduce a potential cellular-based therapy for the prevention of incisional hernia formation. BACKGROUND: Up to 10 % of laparotomies are complicated by postoperative incisional hernias. Despite continuous improvements in surgical technique and technology, hernia rates have remained constant. Cell-based therapies focused on augmentation of the body's natural healing properties could reduce hernia formation. METHODS: Midline laparotomies were performed on 42 Lewis rats. Three groups were studied: (1) primary repair only, (2) primary repair with CollaTape™ (CoTa) overlay and platelet-rich plasma (PRP), and (3) primary repair with CoTa overlay and PRP and bone marrow-derived mesenchymal stromal cells (BM-MSCs). Abdominal wall fascia was recovered at 4 and 8 weeks in each group. Biomechanical testing and histological evaluation was performed. RESULTS: At 4 weeks, there was a twofold increase in tensile strength between groups 1 and 2 and a fourfold increase between groups 1 and 3 (p < 0.001). Group 3 had a 320 % increase in total energy absorption at 4 weeks compared to group 1 and a 142 % increase at 8 weeks (p < 0.001). Vascularization and collagen abundance were significantly increased in group 3 at both time points. CONCLUSION: The addition of BM-MSCs, PRP, and CoTa led to a marked improvement in abdominal wall strength and energy absorption. Histologic evaluation confirmed increased vascularity and collagen abundance consistent with the biomechanical findings. Application of this therapy may ultimately reduce incisional hernia formation.

Potentiation of lipopolysaccharide-induced tumor necrosis factor-alpha expression by 1,25-dihydroxyvitamin D3.

The immunomodulatory hormone 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) has been shown to suppress T-cell proliferation and interleukin-2 synthesis as well as B-cell immunoglobulin synthesis, while stimulating many macrophage functions. We have previously shown increased synthesis of interleukin-1 beta in lipopolysaccharide (LPS)-stimulated U937 cells after pretreatment with 10 nmol/L 1,25-(OH)2D3. We now show that 1,25-(OH)2D3 also primes the increase in U937 cell tumor necrosis factor (TNF)-alpha-accumulated mRNA after activation with LPS; 50% effective concentration (EC50) for the LPS-induced expression of TNF-alpha mRNA was decreased by two orders of magnitude after incubation with 10 nmol/L 1,25-(OH)2D3. Pretreatment of U937 cells with 10 nmol/L 1,25-(OH)2D3 also increased subsequent LPS-induced TNF-alpha mRNA expression by twofold and cell-associated TNF protein levels by more than ninefold. This potentiation was steroid-specific for 1,25-(OH)2D3 because dexamethasone inhibited TNF-alpha mRNA. The potentiation required prior exposure to 1,25-(OH)2D3 for more than 6 hours and was clearly seen after 12 hours. The finding that the sensitivity of the U937 cell monokine response to LPS was dramatically increased by 1,25-(OH)2D3 and the delayed effect on the LPS-stimulated TNF-alpha gene transcript levels indicated that 1,25-(OH)2D3 may be altering the expression of a protein(s) in the U937 cell LPS-signal transduction pathway. In fact, 1,25-(OH)2D3 induced expression of the mRNA for CD14, the high affinity, cell-surface glycoprotein receptor for LPS, which could account for the enhancement of LPS-stimulated monokine gene expression by 1,25-(OH)2D3. Thus, local monokine gene expression may be regulated by both the amount and the temporal entry of the vitamin D hormone and activator(s) into the inflammatory microenvironment.

The human myelomonocytic cell line U-937 as a model for studying alterations in steroid-induced monokine gene expression: marked enhancement of lipopolysaccharide-stimulated interleukin-1 beta messenger RNA levels by 1,25-dihydroxyvitamin D3.

The active metabolite of vitamin D, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], is a potent regulator of human monocyte/macrophage function in vitro. To establish a model for 1,25-(OH)2D3 regulation of human monocyte monokine synthesis, three human cell lines (U-937, THP-1, and HL-60) were examined for: 1) the presence of functional 1,25-(OH)2D3 receptors; 2) the accumulation of interleukin-1 beta (IL-1 beta) mRNA and IL-1 beta protein in response to lipopolysaccharide (LPS); and 3) the regulation of this response by 1,25-(OH)2D3. All three cell lines expressed vitamin D receptor and had increased levels of IL-1 beta mRNA in response to LPS. Preincubation of cells with 1,25-(OH)2D3 augmented IL-1 beta mRNA levels only in U-937 and HL-60 cells. From these data, and taking into consideration their state of differentiation and relative ease of culture, U-937 was chosen over HL-60 and THP-1 as the cell line we further characterized. In U-937 cells, optimum time and dose of pretreatment with 1,25-(OH)2D3 were determined to be 12-24 h at a receptor saturating concentration of 1,25-(OH)2D3 (10 nM). Preincubation of cells with 1,25-(OH)2D3 had no effect on the time course of IL-1 beta mRNA appearance in response to LPS. However, exposure of U-937 cells to 1,25-(OH)2D3 increased by 200% the level of IL-1 beta mRNA detected and decreased by three orders of magnitude the concentration of LPS required to achieve steady state mRNA levels equivalent to those observed in U-937 cells not preincubated with the hormone.2+o

Monoclonal antibodies to immunoglobulin G4 induce histamine release from human basophils in vitro.

Monoclonal murine antibodies to human IgE, IgG1, IgG2, IgG3, IgG4, IgM, and IgA1 were prepared and their abilities to stimulate histamine release from human peripheral blood leukocytes were investigated. As expected, anti-IgE caused 8% to 76% histamine release from the leukocytes of each of 14 nonallergic donors. In one subject anti-IgE alone had no effect, but a 15% histamine release did occur after the subsequent addition of a goat anti-mouse IgG antiserum to crosslink cell-bound anti-IgE molecules. Anti-IgG4 alone caused 5% to 59% histamine release from the cells of four of 14 donors. Subsequent challenge with goat anti-mouse IgG caused an additional release of up to 15% of the basophil histamine, but only from leukocytes of donors responsive to anti-IgG4 alone. No histamine release was detected after stimulation by monoclonal antibodies to IgG1, IgG2, IgG3, IgM, or IgA1. The results of this study indicate that monoclonal antibodies to IgG4 as well as to IgE can stimulate human peripheral blood leukocytes to release histamine. These observations support the theory that antigen-IgG4 interactions can stimulate histamine release from human basophils.