van Marja Luyn

Bone marrow-derived myofibroblasts contribute functionally to scar formation after myocardial infarction.

Myofibroblasts play a major role in scar formation during wound healing after myocardial infarction (MI). Their origin has been thought to be interstitial cardiac fibroblasts. However, the bone marrow (BM) can be a source of myofibroblasts in a number of organs after injury. We have studied the temporal, quantitative and functional role of BM‐derived (BMD) myofibroblasts in myocardial scar formation. MI was induced by permanent coronary artery ligation in mice reconstituted with EGFP or pro‐Col1A2 transgenic BM. In the latter, luciferase and β‐galactosidase transgene expression mirrors that of the endogenous pro‐collagen 1A2 gene, which allows for functional assessment of the recruited cells. After MI, α‐SMA‐positive myofibroblasts and collagen I gradually increased in the infarct area until day 14 and remained constant afterwards. Numerous EGFP‐positive BMD cells were present during the first week post‐MI, and gradually decreased afterwards until day 28. Peak numbers of BMD myofibroblasts, co‐expressing EGFP and α‐SMA, were found on day 7 post‐MI. An average of 21% of the BMD cells in the infarct area were myofibroblasts. These cells constituted up to 24% of all myofibroblasts present. By in vivo IVIS® imaging, BMD myofibroblasts were found to be active for collagen I production and their presence was confined to the infarct area. These results show that BMD myofibroblasts participate actively in scar formation after MI. Copyright

A comparative biocompatibility study of microspheres based on crosslinked dextran or poly(lactic‐co‐glycolic)acid after subcutaneous injection in rats

Microspheres based on methacrylated dextran (dex-MA), dextran derivatized with lactate-hydroxyethyl methacrylate (dex-lactate-HEMA) or derivatized with HEMA (dex-HEMA) were prepared. The microspheres were injected subcutaneously in rats and the effect of the particle size and network characteristics [initial water content and degree of methacrylate substitution (DS)] on the tissue reaction was investigated for 6 weeks. As a control, poly(lactic-co-glycolic)acid (PLGA) microspheres with varying sizes (unsized, smaller than 10 microm, smaller and larger than 20 microm) were injected as well. A mild tissue reaction to the PLGA microspheres was observed, characterized by infiltration of macrophages (MØs) and some granulocytes. Six weeks postinjection, the PLGA microspheres were still present. However, their size was decreased indicating degradation and many spheres had been phagocytosed. The tissue reaction was hardly affected by size differences, except for particles smaller than 10 microm, which induced an extensive tissue reaction. The initial tissue reaction to nondegradable dex-MA microspheres was stronger than towards the PLGA microspheres, but at day 10 the tissue reactions were comparable for both groups. Six weeks postinjection, the dex-MA microspheres were completely phagocytosed, and no signs of degradation were observed. The size and initial water content of dex-MA microspheres hardly affected the tissue response, although less granulocytes were observed for microspheres with higher DS. Slowly degrading dextran microspheres composed of dex-(lactate(1)-)HEMA induced a tissue reaction comparable to the PLGA microspheres. However, degradation of the dex-(lactate(1,3)-)HEMA microspheres was associated with an increased number of MØs and giant cells, both phagocytosing the microspheres and their degradation products. Similar to PLGA, no adverse reactions were observed for the nondegradable dex-MA and degradable dextran microspheres. This study shows that both nondegradable and degradable dextran-based microspheres are well tolerated after subcutaneous injection in rats, which make them interesting candidates as controlled drug delivery systems.

CD34 cells augment endothelial cell differentiation of CD14 endothelial progenitor cells in vitro

Neovascularization by endothelial progenitor cells (EPC) for the treatment of ischaemic diseases has been a topic of intense research. The CD34+ cell is often designated as EPC, because it contributes to repair of ischaemic injuries through neovascularization. However, incorporation of CD34+ cells into the neovasculature is limited, suggesting another role which could be paracrine. CD14+ cells can also differentiate into endothelial cells and contribute to neovascularization. However, the low proliferative capacity of CD14+ cell‐derived endothelial cells hampers their use as therapeutic cells. We made the assumption that an interaction between CD34+ and CD14+ cells augments endothelial differentiation of the CD14+ cells. In vitro, the influence of CD34+ cells on the endothelial differentiation capacity of CD14+ cells was investigated. Endothelial differentiation was analysed by expression of endothelial cell markers CD31, CD144, von Willebrand Factor and endothelial Nitric Oxide Synthase. Furthermore, we assessed proliferative capacity and endothelial cell function of the cells in culture. In monocultures, 63% of the CD14+‐derived cells adopted an endothelial cell phenotype, whereas in CD34+/CD14+ co‐cultures 95% of the cells showed endothelial cell differentiation. Proliferation increased up to 12% in the CD34+/CD14+ co‐cultures compared to both monocultures. CD34‐conditioned medium also increased endothelial differentiation of CD14+ cells. This effect was abrogated by hepatocyte growth factor neutralizing antibodies, but not by interleukin‐8 and monocyte chemoattractant protein‐1 neutralizing antibodies. We show that co‐culturing of CD34+ and CD14+ cells results in a proliferating population of functional endothelial cells, which may be suitable for treatment of ischaemic diseases such as myocardial infarction.

Site-specific tissue inhibitor of metalloproteinase-1 governs the matrix metalloproteinases-dependent degradation of crosslinked collagen scaffolds and is correlated with interleukin-10

We have previously shown that the foreign body reaction (FBR) against crosslinked collagen type I (Col‐I) differs between subcutaneous and epicardial implantation sites; Col‐I was quickly degraded epicardially, whereas degradation was attenuated subcutaneously. The current study set out to dissect the nature and regulation of the MMP‐based degradation of implanted Col‐I in mice during the FBR. Immunohistochemistry showed that MMP‐2, MMP‐8 and MMP‐13 were present in subcutaneous and epicardial implants, whereas only MMP‐9 was also present epicardially. Western blotting showed that MMP‐8 and MMP‐9 were mainly present in their inactive proform. In contrast, collagenase MMP‐13 and gelatinase MMP‐2 were the predominant active MMPs at both sites. Interestingly, the major MMP inhibitor TIMP‐1 was solely observed in subcutaneous implants, which is why MMP‐13 and MMP‐2 are not able to degrade the collagen scaffold at the subcutaneous implantation site. Interleukin 10 (IL‐10), a potent inducer of TIMP‐1 expression, was also mainly detected subcutaneously; giant cells were the main source. Therefore, we surmise that IL‐10, through regulation of the balance between MMPs and TIMP‐1, suppresses the FBR against implanted biomaterials. Together, our findings would provide cues and clues to improve future therapies in regenerative medicine that are based on the tuned regulation of the degradation of biomaterial scaffolds. Copyright

Novel polyurethanes with interconnected porous structure induce in vivo tissue remodeling and accompanied vascularization

Tissue engineering and regenerative medicine have furnished a vast range of modalities to treat either damaged tissue or loss of soft tissue or its function. In most approaches, a temporary porous scaffold is required to support tissue regeneration. The scaffold should be designed such that the turnover synchronizes with tissue remodeling and regeneration at the implant site. Segmented polyester urethanes (PUs) used in this study were based on epsilon-caprolactone (CL) and co-monomers D,L-lactide (D,L-L) and gamma-butyrolactone (BL), and 1,4-butanediisocyanate (BDI). In vitro, the PUs were nontoxic and haemocompatible. To test in vivo biocompatibility, the PUs were further processed into porous structures and subcutaneously implanted in rats for a period up to 21 days. Tissue remodeling and scaffold turnover was associated with a mild tissue response. The tissue response was characterized by extensive vascularization through the interconnected pores, with low numbers of macrophages on the edges and stroma formation inside the pores of the implants. The tissue ingrowth appeared to be related to the extent of microphase separation of the PUs and foam morphology. By day 21, all of the PU implants were highly vascularized, confirming the pores were interconnected. Degradation of P(CL/D,L-L)-PU was observed at this time, whereas the other two PU types remained intact. The robust method reported here of manufacturing and processing, good mechanical properties, and in vivo tissue response of the porous P(CL/D,L-L)-PU and PBCL-PU makes them excellent candidates as biomaterials with an application for soft tissue remodeling, for example, for cardiovascular regeneration.