The foreign body giant cell cannot resorb bone, but dissolves hydroxyapatite like osteoclasts

Foreign body multinucleated giant cells (FBGCs) and osteoclasts share several characteristics, like a common myeloid precursor cell, multinuclearity, expression of tartrate-resistant acid phosphatase (TRAcP) and dendritic cell-specific transmembrane protein (DCSTAMP). However, there is an important difference: osteoclasts form and reside in the vicinity of bone, while FBGCs form only under pathological conditions or at the surface of foreign materials, like medical implants. Despite similarities, an important distinction between these cell types is that osteoclasts can resorb bone, but it is unknown whether FBGCs are capable of such an activity. To investigate this, we differentiated FBGCs and osteoclasts in vitro from their common CD14+ monocyte precursor cells, using different sets of cytokines. Both cell types were cultured on bovine bone slices and analyzed for typical osteoclast features, such as bone resorption, presence of actin rings, formation of a ruffled border, and characteristic gene expression over time. Additionally, both cell types were cultured on a biomimetic hydroxyapatite coating to discriminate between bone resorption and mineral dissolution independent of organic matrix proteolysis. Both cell types differentiated into multinucleated cells on bone, but FBGCs were larger and had a higher number of nuclei compared to osteoclasts. FBGCs were not able to resorb bone, yet they were able to dissolve the mineral fraction of bone at the surface. Remarkably, FBGCs also expressed actin rings, podosome belts and sealing zones-cytoskeletal organization that is considered to be osteoclast- specific. However, they did not form a ruffled border. At the gene expression level, FBGCs and osteoclasts expressed similar levels of mRNAs that are associated with the dissolution of mineral (e.g., anion exchange protein 2 (AE2), carbonic anhydrase 2 (CAII), chloride channel 7 (CIC7), and vacuolar-type H+-ATPase (v-ATPase)), in contrast the matrix degrading enzyme cathepsin K, which was hardly expressed by FBGCs. Functionally, the latter cells were able to dissolve a biomimetic hydroxyapatite coating in vitro, which was blocked by inhibiting v-ATPase enzyme activity. These results show that FBGCs have the capacity to dissolve the mineral phase of bone, similar to osteoclasts. However, they are not able to digest the matrix fraction of bone, likely due to the lack of a ruffled border and cathepsin K

Ultrastructural aspects of foreign body giant cells generated on different substrates

Implantation of biomaterials into the body, e.g. for tissue engineering purposes, induces a material dependent inflammatory response called the foreign body reaction (FBR). A hallmark feature of this response is the formation of large multinucleated cells: foreign body giant cells (FBGCs). Biomaterials like cross-linked and non-cross-linked collagen often induce the formation of FBGCs. It is unknown whether different biomaterials result in the formation of different FBGCs. To investigate this, we implanted cross linked and non-cross-linked dermal sheep collagen subcutaneously in mice. After 21 days the implanted material was collected and prepared for ultrastructural analysis. More FBGCs formed on and between implants of cross-linked collagen compared to non-cross-linked material. The ultrastructural aspects of the FBGCs present on the two types of implants proved to be similar. On both materials, they formed long slender protrusions on the basolateral membrane, they were very rich in mitochondria, contained numerous nuclei, and showed signs of the presence of a clear zone facing the implanted material. Similar clear zones, that resemble osteoclastic features, were also seen in FBGCs generated in vitro on bone slices, but these cells did not form a ruffled border. However, similarities in ultrastructure such as the occurrence of slender protrusions and high mitochondrion content were also found in the FBGCs generated in vitro. These data indicate that FBGCs formed on different substrates share many morphological characteristics. The formation of long finger-like protrusions seemed typical for the FBGCs, in vivo as well as in vitro, however the function of these structures needs further analysis. (C) 2016 Elsevier Inc. All rights reserved

Transmission electron microscopy of osteoclasts and FBGCs.

<p>In the osteoclast cultures, resorption pits were visible with exposed collagen fibrils protruding from the pit surface (<b>a</b>; white arrow). No resorption pits were seen in the FBGC cultures (<b>f;</b> red asterisk = bone). The osteoclasts showed an extensive ruffled border with on both sides a sealing zone (<b>b</b>; red asterisk, <b>c</b>; black arrow). No ruffled border was seen in the FBGCs (<b>f, g</b>). On the basolateral side of the membrane, both cell types showed “finger”-like structures (<b>b, g</b>; black arrow). For FBGCs, these membrane protrusions were generally longer and more abundant than those of osteoclasts. Adjacent to the FBGCs, an electron translucent area was seen covering the bone surface (<b>h</b>; red asterisk), suggesting demineralization. In this area, collagen fibils were exposed (<b>i</b>; white arrow). Both cell types contained high numbers of mitochondria (<b>b, c, g, i</b>; black) and the presence of a sealing zone approximatley 1 μm wide where no organelles were present (<b>e, j</b>; white arrow). Scale bar is 10 μm for panels <b>a</b> and <b>f</b>; 5 μm for panels <b>b</b> and <b>g</b>; 2 μm for panels <b>c</b> and <b>h</b>; and 1 μm for panels <b>d</b>, <b>e</b>, <b>i</b> and <b>j</b>.</p

TRAcP activity of osteoclasts, FBGCs, and macrophages cultured on bone.

<p>Human CD14⁺ monocytes were cultured on bone slices for 25 days with M-CSF and RANK-L (osteoclasts); M-CSF, IL-4, and IL-13 (FBGCs); M-CSF (macrophages) and without cytokines (control). Osteoclasts were TRAcP-positive (<b>a</b>; black arrow) and most contained < 10 nuclei (<b>e</b>). FBGCs were larger with > 10 nuclei (<b>f</b>), and stained less intensely for TRAcP (<b>b</b>; black arrow). Macrophages were generally mononuclear and stained weakly for TRAcP (<b>c, g</b>), similar as CD14⁺ cells that were cultured without cytokines (<b>d, h</b>). Bar plots represent the mean ± S.D. of multinucleated cells (MNCs) per 0.25 cm² bone surface, from 5 independent donors. Scale bar = 100 μm. Red asterisk = bone. *p<0.05, **p<0.01.</p

Osteoclasts and FBGCs cultured on biomimetic hydroxyapatite coatings.

<p>After 25 days of culture, cells were stained for TRAcP activity and nuclei (DAPI). Multinucleated, TRAcP positive osteoclasts (<b>a</b>; black arrow) and FBGCs (<b>b</b>; black arrow) dissolved the coating (coating; red asterisk, plastic; black aterisk). Macrophages were also able to dissolve small parts of the coating (<b>c</b>; black arrow, <b>e</b>). Control wells, incubated without cells, showed no signs of apatite coating dissolution (<b>d</b>). Quantitatively, osteoclasts dissolved more of the coating than the FBGCs (<b>e</b>). Percent dissolution of the hydroxyapatite coating plots represent the mean ± S.D. per 0.32 cm² coating surface. Scale bar = 100 μm. *p<0.05, **p<0.01.</p

qPCR primer sequences.

<p>qPCR primer sequences.</p

Confocal microscopy of osteoclasts and FBGCs.

<p>Plasma membrane (red: CD44 antibody), nuclei (blue: Hoechst nuclei staining), and actin rings (green: phalloidin staining of F-actin) were fluorescently labeled after 25 days culture on bone. Both cell types contained numerous nuclei (<b>a, b, e, f,</b>), actin rings (<b>c, g</b>; white arrow,), and podosome belts (<b>c, g</b>; red arrow). Sagittal views of both cell types composed from the apical side (white asterisk) showed actin structures resembling sealing zones (<b>i, k</b>; white arrows). Sagittal views composed from the basolateral side (red asterisk) of the cells showed round structures composed of actin (<b>j, I</b>; white arrow, red arrow). Scale bar = 50 μm.</p

Resorption activity of osteoclasts and FBGCs on biomimetic hydroxyapatite coatings after concanamycin A treatment.

<p>Cells were cultured for 25 days and stained with acridine orange to visualize sites with low pH. Osteoclasts (left column) and FBGCs (right column) stained positive for acridine orange on both bone (<b>a, f</b>; white dashed circles) and tissue culture plastic (<b>b, g</b>; black dashed circles). After incubation with concanamycin A, acridine orange-positive vacuoles were hardly detected (<b>c, h</b>); moreover, dissolution of hydroxyapatite was blocked (<b>d, I</b>; osteoclasts and FBGCs are visible in black dashed circles) compared to control cells cultured without concamycin A (<b>e, j</b>). Cells were stained for TRAcP and DAPI. Scale bar = 100 μm.</p

Ultrastructural aspects of foreign body giant cells generated on different substrates

Implantation of biomaterials into the body, e.g. for tissue engineering purposes, induces a material-dependent inflammatory response called the foreign body reaction (FBR). A hallmark feature of this response is the formation of large multinucleated cells: foreign body giant cells (FBGCs). Biomaterials like cross-linked and non-cross-linked collagen often induce the formation of FBGCs. It is unknown whether different biomaterials result in the formation of different FBGCs. To investigate this, we implanted cross-linked and non-cross-linked dermal sheep collagen subcutaneously in mice. After 21 days the implanted material was collected and prepared for ultrastructural analysis. More FBGCs formed on and between implants of cross-linked collagen compared to non-cross-linked material. The ultrastructural aspects of the FBGCs present on the two types of implants proved to be similar. On both materials, they formed long slender protrusions on the basolateral membrane, they were very rich in mitochondria, contained numerous nuclei, and showed signs of the presence of a clear zone facing the implanted material. Similar clear zones, that resemble osteoclastic features, were also seen in FBGCs generated in vitro on bone slices, but these cells did not form a ruffled border. However, similarities in ultrastructure such as the occurrence of slender protrusions and high mitochondrion content were also found in the FBGCs generated in vitro. These data indicate that FBGCs formed on different substrates share many morphological characteristics. The formation of long finger-like protrusions seemed typical for the FBGCs, in vivo as well as in vitro, however the function of these structures needs further analysis

Aortic coarctation repair through left thoracotomy: Results in the modern era

OBJECTIVES: Surgical repair of coarctation of the aorta (CoA) is often possible through left thoracotomy and without the use of cardiopulmonary bypass. Recent studies reporting the outcome after CoA repair through left thoracotomy are limited. Therefore, the aim of this study is to evaluate the results of CoA repair through left thoracotomy in children who were operated on in our centre over the past 21 years. METHODS: From January 1995 to December 2016, 292 patients younger than 18 years underwent primary CoA repair through left thoracotomy at our 2 institutions. Peri- and postoperative data and follow-up data collected from our hospital and the referring hospitals were retrospectively reviewed. RESULTS: Median age at operation was 64 days (range 2 days-17 years). Most patients underwent the resection of the CoA followed by an (extended) end-to-end anastomosis (93%). Six patients died perioperatively and 2 more patients died during the follow-up, of which 7 patients had other major comorbidities. Actuarial survival was 97% at 5 years, 96% at 10 years and 96% at 15 years. Second arch interventions due to recoarctation were performed in 9.9% (n = 29) of patients, consisting of balloon dilatation in all but 2 patients. Recoarctation occurred significantly more often after initial repair in the neonatal period (21%) and could occur as late as 14 years after initial surgery. There were 7 re-recoarctations, and 14% of patients were on hypertensive medication during the follow-up. CONCLUSIONS: Repair of CoA through left thoracotomy is a safe procedure with low rates of mortality. The long-term follow-up is necessary due to the significant risk of recoarctation requiring reintervention