Poly(methacrylic acid -co- methyl methacrylate) beads were previously shown to have a therapeutic effect on wound closure through the promotion of angiogenesis. However, it was unclear how this polymer elicited its beneficial properties. The goal of this thesis was to characterize the host response to MAA beads by identifying molecules of interest involved in MAA-mediated angiogenesis (in comparison to poly(methyl methacrylate) beads, PMMA). Using a model of diabetic wound healing and a macrophage-like cell line (dTHP-1), eight molecules of interest were identified in the host response to MAA beads. Gene and/or protein expression analysis showed that MAA beads increased the expression of Shh, IL-1β, IL-6, TNF-α and Spry2, but decreased the expression of CXCL10 and CXCL12, compared to PMMA and no beads. MAA beads also appeared to modulate the expression of OPN. In vivo, the global gene expression of OPN was increased in wounds treated with MAA beads, compared to PMMA and no beads. In contrast, dTHP-1 decreased OPN gene expression compared to PMMA and no beads, but expressed the same amount of secreted OPN, suggesting that the cells decreased the expression of the intracellular isoform of OPN. Interestingly, MAA beads had no effect on the expression of pro-angiogenic growth factors VEGF, bFGF and PDGF-B in vivo or in vitro, suggesting that MAA beads do not induce angiogenesis by simply increasing the expression of pro-angiogenic factors, but use more subtle mechanisms. It was hypothesized that these mechanisms may involve modulation of toll-like receptor signaling in macrophages interacting with the protein layer adsorbed on to MAA beads, in a manner distinct from PMMA beads and no beads. Taken together, the results suggest that MAA beads promote angiogenesis through increased expression of Shh, decreased expression of CXCL10 and modulation of the expression of OPN, but not through increased expression of typical pro-angiogenic growth factors. The resulting vessel-rich “alternative foreign body reaction” has exciting clinical implications as the polymer itself was found to exert a therapeutic effect in the absence of bioactive components or transplanted cells. Understanding the mechanism could lead to new applications for this material and others designed on similar principles.

The aim of this study was to investigate the effects of various polysaccharides and their breakdown products on the proliferation and migration of cells involved in wound healing, both in vitro and in vivo, with the ultimate aim of developing a commercially viable collagen dressing containing an active polysaccharide fragment which would stimulate the wound healing response to such a degree that good quality and significantly faster healing would take place. Hyaluronic acid (HA), chondroitin sulphate (CS), heparin, Oxidised Regenerated Cellulose (ORC) and pectin were tested in this study. Some HA fragments and CS fragments significantly stimulated (p<O.05) the proliferation of Bovine Aortic Endothelial (BAEC) cells, although other HA or CS fragments were without effect. All HA and CS fragments tested also had no effect on the migration of L929 cells in the Boyden Chamber assay. Pectin stimulated the proliferation and migration of L929 cells, whereas, ORC 1 and heparin both suppressed proliferation (25% - 45% inhibition) of these cells but stimulated their migration. When injected subcutaneously into Polyvinyl alcohol sponges in the rat wound model at concentrations of 10 and 50J.1g/ml, heparin brought about an increased presence of granulation tissue in the wound after 10 days and 7 days respectively. These results indicate that structurally similar polysaccharides can have profoundly different effects on cell proliferation and migration, and thus have potential therapeutic use in guiding cell movement in wound healing.

No description available.

名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成25年3月25日 原真由氏の博士論文として提出された

Hypothesis: Trimethoprim promotes wound healing, decreases lesion counts and improves quality of life of recessive dystrophic epidermolysis bullosa (RDEB) patients. Objectives: Assess feasibility of conducting a large randomized clinical trial. Determine efficacy of trimethoprim in healing of chronic wounds, decreasing lesion counts and improving quality of life of RDEB patients. Methods: Prospective, randomized, double-blinded, placebo-controlled, cross-over pilot study. Results: Ten patients enrolled in the trial, 7 completed both study periods. Despite showing that all patients improved on trimethoprim and that there was a 41% difference in affected area percent change favoring trimethoprim, the cross-over analysis did not show a significant difference between the drug and placebo (p=0.08). Secondary outcome measures did not achieve statistical significance. Limitations: Small sample size, large variation in wound size and unaccounted confounders. Conclusions: Although patients experienced improvement while on trimethoprim, no statistical significant change was showed when compared to placebo.

Potential targeting of impaired wounds resulting from hyperglycemia using mesenchymal stem cells is a promising approach. We hypothesized that when administered to dermal wounds in hyperglycemic subjects, neonatal mesenchymal stem cells (MSCs) would be more effective than adult mesenchymal cells in accelerating healing. We examined the in vitro impact of various glucose conditions on proliferation and senescence of human umbilical cord perivascular cells (HUCPVCs) and adult bone marrow MSCs (hBM-MSCs). We also investigated the healing potential of both cells in dermal wounds of streptozotocin-induced NOD-scid-gamma (NSG) mice utilizing planimetry and histomorphometry. HUCPVCs showed higher proliferation under normal and hyperglycemic conditions and lower senescence under all conditions compared to hBM-MSCs. Wound closure was better in treated wounds compared to untreated wounds. Disease tolerance varied among mice which affected healing. HUCPVCs still holds a potential over adult MSCs for impaired wounds; yet more studies are needed to recognize their bona fide capacity.

Hypothesis: Trimethoprim promotes wound healing, decreases lesion counts and improves quality of life of recessive dystrophic epidermolysis bullosa (RDEB) patients. Objectives: Assess feasibility of conducting a large randomized clinical trial. Determine efficacy of trimethoprim in healing of chronic wounds, decreasing lesion counts and improving quality of life of RDEB patients. Methods: Prospective, randomized, double-blinded, placebo-controlled, cross-over pilot study. Results: Ten patients enrolled in the trial, 7 completed both study periods. Despite showing that all patients improved on trimethoprim and that there was a 41% difference in affected area percent change favoring trimethoprim, the cross-over analysis did not show a significant difference between the drug and placebo (p=0.08). Secondary outcome measures did not achieve statistical significance. Limitations: Small sample size, large variation in wound size and unaccounted confounders. Conclusions: Although patients experienced improvement while on trimethoprim, no statistical significant change was showed when compared to placebo.

Potential targeting of impaired wounds resulting from hyperglycemia using mesenchymal stem cells is a promising approach. We hypothesized that when administered to dermal wounds in hyperglycemic subjects, neonatal mesenchymal stem cells (MSCs) would be more effective than adult mesenchymal cells in accelerating healing. We examined the in vitro impact of various glucose conditions on proliferation and senescence of human umbilical cord perivascular cells (HUCPVCs) and adult bone marrow MSCs (hBM-MSCs). We also investigated the healing potential of both cells in dermal wounds of streptozotocin-induced NOD-scid-gamma (NSG) mice utilizing planimetry and histomorphometry. HUCPVCs showed higher proliferation under normal and hyperglycemic conditions and lower senescence under all conditions compared to hBM-MSCs. Wound closure was better in treated wounds compared to untreated wounds. Disease tolerance varied among mice which affected healing. HUCPVCs still holds a potential over adult MSCs for impaired wounds; yet more studies are needed to recognize their bona fide capacity.

Patients who suffer from severe joint destruction caused by arthritis often undergo total joint arthroplasty (TJA). A major limitation of this treatment and common long-term complication is the development of aseptic loosening of the prosthesis in as many as 20% of patients. The current paradigm to explain aseptic loosening proposes that wear debris generated from the prosthesis initiates a macrophage-mediated inflammatory response by resident macrophages, leading to osteoclast activation and bone resorption at the implant interface. This can then lead to the development of a peri-prosthetic fracture. The principal aim of fracture healing is to restore the bone to its original form and strength. However, this ultimate goal can be altered if the healing is impaired. This impairment may be due to bone disease (osteoporosis) or even the introduction of a foreign material such as PE wear debris that could have migrated from the articulating surface to the fracture site. A standard closed unilateral fracture of the right femur was performed in both normal and oestrogen deficient rats following fixation with a k-wire. Ceridust (PE wear debris) was combined with hyaluronic acid and saline and injected directly into the fracture site. Femurs were assessed using radiographs, histology and immunohistochemistry. Histological analysis revealed that complete remodelling was achieved in all control groups by 6 weeks post-fracture with mechanical strength returning to normal values. The mechanical properties of the fractures were not influenced by the presence of PE wear debris in the dose and timing examined. Histology and immunohistochemistry however, did reveal a local effect of the presence of PE wear debris. The histology adjacent to the PE particles was inferior to the controls but did not manifest itself in a reduction in the mechanical properties except in the oestrogen deficient bone at 6 weeks post-fracture. The levels of MMP-1 and TNF-?? correlated to the presence of PE particles. In this thesis, I have shown the mechanism by which bone remodelling in fracture healing could be retarded due to the presence of PE wear debris, by increased matrix degradation in both normal and oestrogen deficient animals.

Transglutaminase II (TG2) is a complex protein with five different reported activities. Increases in TG2 expression and TGase activity have previously been observed during wound healing in rat studies; however, it has been unclear whether these phenomena were directly involved in the healing process or if they were simply a by-product of it. The aims of this thesis were, thus, to determine if TG2 plays a role in wound healing in vivo and to elucidate the mechanism of any effects TG2 may have at the cellular level. TG2 ablation resulted in delayed wound healing. To gain mechanistic insight into this abnormality, primary fibroblast cultures from TG2-knockout and wildtype mouse embryos were analysed. TG2-null fibroblasts displayed decreased adhesion and integrin signalling during initial stages of adhesion. Intriguingly, TG2-null cells showed faster activation of Rac1 and RhoA in response to adhesion. Long-term adhesion of TG2-null fibroblasts resulted in increased basal phosphorylation of FAK and number of paxillin-stained focal adhesions, enhanced PI3-kinase signalling, faster actin dynamics and altered activation of p44/42 MAPK. These results are indicative of futile cycling of intracellular signalling pathways resulting from reduced focal adhesion turnover in the TG2-knockout fibroblasts. Rescue experiments demonstrated that TG2-mediated effects on cell adhesion occurred in the extracellular environment and that neither GTP-binding nor TGase activity is required for these effects. Results further showed that a ???compact??? conformation of TG2 was not required for this role of TG2. Interestingly, addition of recombinant TG2 to the extracellular environment increased cell spreading of TG2-null cells to a level far greater than that seen in wildtype cells, which did not increase their spreading in response to exogenous TG2. Demonstration of faster activation of the small GTPases in the TG2-null MEFs, and the apparent inhibition of exogenous TG2???s extracellular effects on cell spreading by endogenous protein in the wildtype cells, provide tantalising evidence for a role for intracellular TG2 in regulating activation of the small GTPases to promote efficient fibroblast migration. This work identifies TG2 as a facilitator of efficient wound closure through extracellular effects on integrin-mediated signalling and intracellular effects on activation of the small GTPases.