Skip Navigation


European Heart Journal Advance Access originally published online on January 23, 2007
European Heart Journal 2007 28(11):1389-1396; doi:10.1093/eurheartj/ehl422
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
28/11/1389    most recent
ehl422v1
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (2)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by De Visscher, G.
Right arrow Articles by Flameng, W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by De Visscher, G.
Right arrow Articles by Flameng, W.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The European Society of Cardiology 2007. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

In vivo cellularization of a cross-linked matrix by intraperitoneal implantation: a new tool in heart valve tissue engineering

Geofrey De Visscher*, Ilse Vranken, An Lebacq, Christiaan Van Kerrebroeck, Javier Ganame, Erik Verbeken and Willem Flameng

Laboratory for Experimental Cardiac Surgery, Department of Cardiovascular Diseases, Katholieke Universiteit Leuven, Minderbroedersstraat 17, 3000 Leuven, Belgium

Received 21 April 2006; revised 26 October 2006; accepted 10 November 2006; online publish-ahead-of-print 23 January 2007.

* Corresponding author. Tel: +32 16 33 71 20; fax: +32 16 33 71 22. E-mail address: geofrey.devisscher{at}med.kuleuven.be


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 
Aim: To use in vivo instead of in vitro cell seeding in heart valve tissue engineering.

Methods and results: Intraperitoneally preseeded, photo-oxidized bovine pericardial pulmonary valve constructs (group 1) were compared with non-preseeded constructs (group 2) implanted in sheep. All valves functioned normally and were macroscopically intact at explantation [1 week (n = 6) and 1 month (n = 6) in each group], except for one thrombosed leaflet in a group-2 valve at 1 month. Almost 10-fold higher neomatrix deposition and doubling of the leaflet thickness were found in group 1 vs. 2 (P < 0.05). A concomitant significant decrease in leaflet length (15%) was found at 1 month in group 1. The total cross-sectional surface and total amount of collagen of the original matrix remained unchanged in both groups at all times. Immunohistochemistry showed a low immune response, stem/progenitor cell infiltration, appropriated differentiation, and spontaneous endothelialization of the valves. Significantly, increased re-cellularization was found after IP preseeding compared with spontaneous seeding: cell coverage of the leaflet was 71–100 vs. 8–26% (P < 0.05), respectively.

Conclusion: Complete re-cellularization can be obtained by IP preseeding of an acellularized cross-linked matrix. Well-functioning valve constructs show cellularization and differentiation into myofibroblast phenotype and concomitant neomatrix deposition.

Key Words: Tissue engineering • Endogenous seeding • Heart valve

This paper was guest edited by Prof. Julie H. Campbell, University of Queensland, Australia


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Acknowledgements
 References
 
The disadvantages of actual mechanical and bioprosthetic heart valves are related to thromboembolic and bleeding complications as well as to calcification or degenerative valve dysfunction.1 Recent attempts to overcome these problems by tissue engineering of heart valves focus on the technology of in vitro seeding of host cells on biodegradable synthetic scaffolds or non-cross-linked acellularized matrices.2 Although some experimental success was claimed, durability of these constructs in terms of the absence of calcification and failure of the prosthetic valve remains to be proved. Furthermore, permanent and complete re-cellularization of the valve structures protecting the valve against degeneration and thrombosis was never demonstrated.

The reasons for these flaws is basically the inherent potential of the scaffold to degenerate and the need for a permanent and complete repopulation of the construct by highly differentiated valve cells capable of producing a new matrix material having natural biophysical properties.

Although it must be theoretically possible to generate a functional heart valve using the concept of in vitro cell seeding on a degradable matrix, we preferred a completely different approach. At first, we used a stable, cross-linked matrix that would not be degraded over time, to guarantee a durable heart valve, having adequate biophysical properties and lasting for a long time without degradation. Second, we applied the concept of endogenous cell seeding using the natural ‘milieu interior’ for attraction, homing, and differentiation of progenitor cells, in contrast to the artificial and limited in vitro seeding conditions. We used the organism as its own bioreactor, based on the work of Campbell et al.,3 who generated a functional vascular graft from an intraperitoneally (IP) deposited capsule around an implanted silastic tube. The capsule comprises an internal layer of macrophages, some layers of (myo)fibroblasts and an external layer of mesothelial cells and is generally described as ‘foreign body reaction’.4 By studying the cell morphology and co-localization of alpha smooth muscle actin (ASMA) and CD172a, it has been proven that the entire layer of (myo)fibroblasts is of haematopoietic origin5 and that the myofibroblasts in those conditions were derived from macrophages.6 On the other hand, the presence of an undifferentiated cell population in the explants was also reported.6 ASMA is a generally used marker for myofibroblasts,7 but CD172a is not an exclusive marker for macrophages and is also expressed in a subpopulation of haematopoietic stem cells.8,9 Therefore, the possible presence of stem cells in granulation tissue renders the study of the ‘early’ foreign body reaction as a means to repopulate a biological but stabilized matrix attractive. Based on the presence of approximately 4.5% lineage negative cells on 3 day IP implants in rats (unpublished results), we performed 3 day endogenous IP seeding by the valve recipient itself for this study. As a cross-linked scaffold we have used a non-cytotoxic photo-oxidized bovine pericardium,10 which is shown to tolerate in vitro cell seeding.11

The aim of this study was primarily to prove complete re-cellularization of the scaffolds and to demonstrate appropriate cell differentiation and phenotype. On the other hand, we also wanted to show that the valves remain haemodynamically stable up to 1 month after implantation in the pulmonary position, free of thrombosis in the absence of anti-coagulation therapy and free of calcification.

At present, it was not our intention to compare this valve to any type of existing bioprostheses in terms of durability because the actual design of the valve is experimental and meant only as a tool to prove re-cellularization potential.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Acknowledgements
 References
 
Animals and study design
Lovenaar sheep [n = 24, ewes 60 kg (45–75)] were selected and cared for in accordance with the ‘Guide for the Care and Use of Laboratory Animals’ (NIH publication 85-23, revised 1985). The study was approved by the local Ethics Committee.

Photo-oxidized (CardiofixTM, Sulzer Carbomedics, TX, USA) pericardium was used.

The study comprised four groups to which the sheep were randomly assigned and the groups contained six animals each: group 1, spontaneous seeding and 1 week in pulmonary position; group 2, spontaneous seeding and 1 month in pulmonary position; group 3, IP preseeding and 1 week in pulmonary position; and group 4, IP preseeding and 1 month in pulmonary position. The sheep of groups 3 and 4 were implanted in the peritoneal cavity with a pericardial patch suspended in a stainless steel cage. Three days later, the implant was aseptically retrieved and a valve (Figure 1) was made, similar to the one described by White et al.12 In short, a 6.8 x 3.8 cm pericardium patch was folded along the long axis leaving an extra 2 mm at on end. The fold was fixed with one 5-0 prolene suture at each side, the whole flap was folded along the short axis, and the short axes were sewn together with a running suture. The double-walled tube was suspended on a large forceps with the suture on one of the tips. At two-thirds of the length, distal from the sutured edge, one suture was used to create a leaflet and the same was done on the other side of the double-walled tube. A left thoracotomy was performed, cardio-pulmonary bypass installed, the native pulmonary valve excised, and the in vivo seeded valvular construct implanted as an interposition in the pulmonary artery. The valves were retrieved after 1 week or 1 month in pulmonary position for groups 3 and 4, respectively. The sheep of groups 1 and 2 only received the valve without prior IP seeding. Similarly, the valves were retrieved after 1 week or 1 month for groups 1 and 2, respectively.


Figure 1
View larger version (112K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 1 Finalised 21 mm diameter valve construct ready for implantation.

 
Procedure for ovine implant echocardiography
At sacrifice, 7 or 31 days postoperatively, the animals were first premedicated with ketamine and anaesthesia was induced with isoflurane in oxygen. The animal was then placed on the operation table in right lateral recumbent position and attached to the anaesthesia machine. The echocardiography was performed by an experienced medical doctor using a Vivid Five echo system (GE medical systems) and a 2.5 MHz GE Ultrasound probe. Prior to excision of the valve, the animal was heparinized to prevent blood clotting and sacrificed with an overdose of pentobarbital and KCl.

Histology and immunohistochemistry
After overnight fixation in 4% paraformaldehyde, the samples were incubated overnight in 20% sucrose in PBS. The samples were snap-frozen in Neg-50 medium (Prosan, Belgium) and stored at –80°C. Seven micrometre cryosections of the samples were placed on poly-L-lysine-coated slides and stored at –20°C until staining.

Cross-linked matrix stability assessment was performed by means of picrosirius red staining13 allowing measurement of the total collagen as well as the organized collagen (Figure 2). We measured the total area of the pericardium (At, between green lines) and the area of the red stained material within that area (Ar). An additional picture was taken using polarized light to measure the organization of the collagen. To obtain this picture, the section was placed in between two perpendicularly orientated polarization filters, the collagen bundles within the section themselves also act as polarization filters thus causing these bundles to be visible again. On this image, the area of the red illuminated bundles (Ab) is measured. The collagen density and organized collagen are calculated by the following formulas.

  1. Collagen density: (Ar/At) x 100
  2. Organized collagen: (Ab/Ar) x 100


Figure 2
View larger version (80K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 2 Microscopy images of picrosirius red stained photo-oxidized bovine pericardium with clear field illumination (upper left) and polarized illumination (upper right). The lower panels are representative micrographs of each group. Spontaneous, non-IP-seeded valve group. Each individual micrograph is showing the picrosirius red stained collagen and in blue pseudo-color the organized collagen bundles photographed with polarized light.

 
Both are expressed as a percentage: collagen density as a percentage of the total area and organized collagen as a percentage of the collagen area.

This method allows studying the matrix integrity both on a microscopic and molecular organization level. On the microscopic level, it allows assessment of gross structural damages such as lacerations and increase or decrease in spongiosity. On the molecular organization level, it allows the study of bundle breakdown, an important feature that might occur during cellular infiltration.

Before immunohistochemistry (IHC) was performed, the presence of cells was verified using standard haematoxylin and eosin (H&E) staining and microscopic screening. Additional MOVAT pentachrome staining was performed to evaluate the composition of connective tissue elements in the newly deposited matrix.13

The samples were immunohistochemically stained for the following markers: CD45 (clone: 1.11.32, Serotec, Oxford, UK), CD44 (clone: BAT31A, A&E Scientific SPRL, Marcq, Belgium); major histocompatibility complex (MHC)-I (clone: H58A, A&E Scientific SPRL, Marcq, Belgium); MHC-II (clone: TH14B, A&E Scientific SPRL, Marcq, Belgium); phosphohistone H3 [rabbit polyclonal (06-570 Upstate), Biognost, Heule Belgium]; vimentin (clone: V9, DAKO, Heverlee, Belgium); ASMA (clone: 1A4, DAKO, Heverlee, Belgium); smooth muscle heavy chain (SMMS-1, clone: SMMS-1, DAKO, Heverlee, Belgium); CD172a (clone: DH59B, A&E Scientific SPRL, Marcq, Belgium); CD34 (clone QBEnd10, DAKO, Heverlee, Belgium); CD117 [rabbit polyclonal (ab5633), Abcam, Cambridge, UK]; and endothelial nitric oxide synthase (ecNOS, clone: 3, BD Pharmingen, Erembodegem, Belgium). The cells from one complete leaflet section were counted for each valve, the length of the leaflet was measured as well as the thickness and surface of the deposited new material. Length, thickness, and surface area of the neomatrix were measured on calibrated micrographs by image analysis, using an Axioplan 2 imaging microscope (Zeiss, Belgium) and the Axiovision 4.3 software package (Zeiss, Belgium). For neomatrix surface calculation, the surface of the original matrix was subtracted from the total leaflet surface, both measured on the same picrosirius red stained section. For cellular phenotyping, a total of 500 cells were assessed for each leaflet and the results were expressed as a percentage. To avoid bias, several pictures from each leaflet were taken, divided into quarters, and counted according to a randomization list.

Statistical analysis
The sample size of the groups was based on previous experiences and in accordance, non-parametric statistics were used. A first analysis was performed by means of a two-tailed Kruskal–Wallis test for n independent samples (SPSS 11, SPSS Inc., Chicago, IL, USA) for continuous data or Pearson's {chi}2 test for the ordinal data. P-values less than or equal to 0.10 were considered significant and additional testing was only performed for these groups. For continuous data, exact non-paired two-tailed Wilcoxon–Mann–Whitney tests were used to identify the differences between the individual groups and P-values less than 0.05 were regarded as significant. For the ordinal data, Pearson's {chi}2 test was used and P-values less than 0.05 were regarded as significant. Data were only compared when the groups were either within the same seeding protocol or within the same time frame. Since the IP test samples were only primed with cells, the observed fractions of different cells were only compared with the valves made out of this material.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Acknowledgements
 References
 
Functionality of the ovine valve implants
As shown in Table 1, all animals were within the same range of age and weight, a requirement for implantation of a 21 mm diameter valve construct. As shown in the same table, all except one valve construct were adequately implanted. In one valve, one leaflet was caught by the distal running suture of the end-to-end anastomosis with the native pulmonary artery, resulting in one small and two large leaflets. Assessment of the haemodynamic function was performed but showed no differences between any of the groups. Equal gradients and pulmonary insufficiency scores were observed. Regarding thrombus deposition, none of the six non-preseeded valves, explanted at 1 week, showed valve thrombosis. At 1 month, however, one out of six valves presented an obstructive thrombus. All preseeded valves were free of thrombosis at 1 week and at 1 month.


View this table:
[in this window]
[in a new window]

  		Table 1 Age, weight, and valve function

 
Re-cellularization and remodelling of the ovine valve implants
A four- to seven-fold higher cell count was found in the IP-seeded group as compared with the spontaneously seeded group, at 1 week and 1 month, respectively (Table 2 and Figure 3; H&E stain). In and on spontaneously seeded valve leaflets, cellularization remained constant. Although a further numerical increase in cell count was apparent in the IP-preseeded valves at 1 month, this increase did not reach a statistical significance.


View this table:
[in this window]
[in a new window]

  		Table 2 Image analysis and matrix stability data

 

Figure 3
View larger version (38K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 3 Representative H&E and MOVAT stained sections of valves from each group. Spontaneous, non-intraperitoneally seeded valve group.

 
Cell density and the amount of leaflet covered by the new material were estimated, as shown in Figure 4. A significantly higher cell density was found in the IP-preseeded groups as compared with the spontaneously seeded groups (upper panel). A similar result was found regarding the coverage of the implanted material with newly deposited material (lower panel). Figure 3 shows H&E or MOVAT pentachrome stained representative sections of both control and IP-preseeded valves after 1 week or 1 month in the pulmonary position.


Figure 4
View larger version (15K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 4 (A) Cell density data [median (interquartile range)] from the spontaneously seeded and IP-preseeded photo-oxidized valve constructs implanted in the pulmonary position for either 1 week or 1 month. (B) Leaflet coverage [(length of newly deposited material/leaflet length) x 100] from the spontaneously seeded and IP-preseeded photo-oxidized valve constructs implanted in the pulmonary artery for either 1 week or 1 month.

 
The IP-preseeded leaflets showed a significant 15% shortening after being 1 month in the pulmonary position. Leaflet thickness of IP-preseeded valves is twice that of the spontaneously seeded valves. De novo deposited or neomatrix surface is approximately 10-fold larger when comparing IP-preseeded with spontaneously seeded valves (Table 2 and Figure 3; MOVAT stain). Due to one outlier in the spontaneously seeded valves at 1 week after implantation, statistical significance between both 1 week groups could not be attained. With respect to the new matrix deposition, we found that collagens were present (picrosirius red, data not shown), but that organization into bundles was not yet achieved. The blue-green staining of the new matrix material also revealed the presence of ground substance and proteoglycans.

Matrix stability
Table 2 also summarizes the matrix stability data. No differences in collagen density could be observed. All these data were within the range of collagen density observed in the test samples, which were only implanted in the peritoneal cavity for 3 days [84.1 (73.2, 90.1)%]. Regarding the percentage of organized collagen, we found a limited but significant decrease in collagen organization in the spontaneously seeded implants in the 1 month group as compared with the 1 week group. This decrease was already apparent in the 1 week IP-preseeded group but did not change with time. Additionally, we also found that this decreased collagen organization was already present after 3 days of IP implantation [90.3 (77.6, 94.8)%].

Cell phenotyping in valve constructs
Table 3 summarizes the immunohistochemical cellular phenotyping of spontaneously seeded and IP-preseeded valves, and IP test sample implanted in the sheep's peritoneal cavity for 3 days. Since these are percentages, only the fraction of positive cells is shown, keeping in mind the large differences in total cells observed, large differences in the absolute cell type count are apparent. CD44+ cells, absent in IP test samples, showed a significant increase from 1 week to 1 month in spontaneously seeded valves only. Expression of MHC-I and -II was low. At 1 week, MHC-I expression significantly differed between spontaneously seeded and IP-preseeded valves. Phosphohistone H3 showed a low but consistent presence of dividing cells. In the spontaneously seeded constructs, ASMA+ or vimentin+ cells increased significantly from 1 week to 1 month to levels already attained by IP-preseeded valves at 1 week. At both time points, there was an insignificant presence of SMMS-1+ cells. CD34+ cells were present in low percentages in all groups but not in all individual valves. CD117+ cells, absent in 1 week spontaneously seeded valves, increased significantly at 1 month, to the level already attained by IP-preseeded valves at 1 week of pulmonary implantation.


View this table:
[in this window]
[in a new window]

  		Table 3 Immunohistochemical data

 
We realize that the valve tissue treatment as we performed is not optimal for maintaining the endothelium: ideally the valves should have been fixed in situ with paraformaldehyde. However, the initial H&E stains revealed that we could have an endothelial cell layer, which we stained for endothelial NO synthase (ecNOS). Figure 5 shows different aspects of the re-cellularization. Panels A–C show the stainings for vimentin, ASMA, and SMMS-1, respectively. These micrographs illustrate the presence of blast-like and spindle-shaped vimentin+ cells (A), ASMA+ spindle-shaped cells, and only limited presence of SMMS-1+ cells. Panels D and E illustrate the presence of CD117+ or CD34+ cells, respectively. The DAPI-stained section in panel F shows completely re-cellularized photo-oxidized bovine pericardium. Panel G shows an incompletely organized endothelial layer on an IP-seeded valve construct that was implanted in the pulmonary artery for 1 week. However, at 1 month we found organized endothelial layers on the wall and fibrosa side of the leaflet (H) and on both sides of the leaflet (I). Presence of endothelium (ecNOS) was scored as present (1) or absent (0) and summarized in Table 3. At 1 week, IP-preseeded valves had a significantly higher proportion of endothelialized valves than the spontaneously seeded valves. There was also a significant increase in endothelialization when compared with the IP test samples, which were completely devoid of any endothelial cell.


Figure 5
View larger version (92K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 5 (A) Vimentin+ cells (red) with blast-like morphology (outer layer) and spindle shape (interior layer) in IP-preseeded valves after 1 month in the pulmonary position. (B) ASMA+ cells, mainly spindle-shaped cells, in the interior layer in IP-preseeded valves after 1 month in the pulmonary position. (C) Smooth muscle heavy chain myosin positive cells. (D) CD117+ cells on an IP-preseeded valve after 1 week in the pulmonary position. (E) CD34+ cells on an IP-preseeded valve after 1 week in the pulmonary position (CD34, green; nuclei, blue). (F) Completely re-cellularized photo-oxidized matrix (nuclei, blue). (G) One-week-old unorganized endothelial layer (ecNOS, green; nuclei, blue). (H) One-month-old organized endothelial layer on the wall (left) and the fibrosa side (right) of the leaflet (ecNOS, green; nuclei, blue). (I) One-month-old organized endothelial layer on the fibrosa (left) and ventricularis side (right) of the leaflet (ecNOS, green; nuclei, blue).

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Acknowledgements
 References
 
To provide evidence that the combination of photo-oxidized bovine pericardium and cells seeded by the short IP implantation is suitable as heart valve prosthesis, we implanted this material in the pulmonary valve position and compared them to valves made from the same material but without IP cell seeding. Despite the fact that the valves were handcrafted and therefore prone to variations, no abnormal valve function could be detected. This was independent of the treatment of the valves, that is, IP-preseeded or not. Only the absence of valve thrombosis suggests the superiority of the IP-preseeded valves.

Re-cellularization was clearly dependent upon prior IP seeding of the matrix for three days. IP-preseeding covered the valves almost completely with new tissue containing more cells and new extracellular matrix than could be achieved by spontaneous seeding of similar constructs. On the other hand, an average 15% shrinkage of the leaflets was observed in IP-seeded valves after being 1 month in the circulation. The actual valves were designed to assure adequate leaflet coaptation in spite of this 15% shrinkage. Since cells have infiltrated the original matrix, it was important to look at the structural integrity of the matrix. No decrease in the amount of collagen was observed, only a small decrease in organization. However, this was coinciding with the repopulation of the material and can be caused by the cells growing and migrating in between the collagen fibres, inducing a limited dissociation of the collagen structure.

Cells having a role in inflammation and the immune response are present in low percentages. It is interesting that in spontaneously seeded valves cells positive for CD44, a receptor for hyaluronic acid14 involved in homing, of among others, primitive cells,15 increase three times from 1 week to 1 month after implantation in the pulmonary artery and are three times higher than the CD45+ cells, a panleukocytic marker.16 The IP test samples were completely devoid of CD44+ cells, whereas the IP-preseeded valves had a significantly larger proportion of these cells. This suggests either a differentiation of these cells or an attraction of cells from the blood. In this respect it is also noteworthy to mention the existence of CD45–/CD44+ cells, of bone marrow stromal origin, also expressing markers like Stro-1.17

The low presence or even absence of antigen presenting molecules MHC-I and -II also shows that the material is only minimally immunogenic.18 To define whether we have a proliferative tissue, we assessed the proliferation rate of the cells by staining for phosphohistone H3.19 In general, we found a low mitotic index (around 1%) in all valves, which was not different from the mitotic index of the IP test samples. It is known that in foreign body reaction the cells are rather attracted to the material than proliferating in situ.4,20,21 Furthermore, mitotic indices of 10–20% are necessary for cardiac muscle regeneration,22 whereas insufficient myocardial repair in normal mice relies on cell attraction.23

Since the method of IP preseeding is based on the attraction of immature cells, we have also studied their differentiation into (myo)fibroblasts and smooth muscle cells, the original heart valve interstitial cells.7 Three markers were used:1 vimentin, specific for mesenchymal cells including fibroblasts;2 ASMA, found in myofibroblasts and smooth muscle cells; and3 heavy chain myosin, specifically found in smooth and skeletal muscle cells.7,24 From this phenotyping, we can conclude that a dramatic differentiation of the cells occurs when we compare the 1 week to the 1 month spontaneously seeded group: there is a six-fold increase in the amount of myofibroblasts. The IP test samples were already primed with cells of mesenchymal origin and this resulted in a differentiation of the cells after being implanted in the pulmonary artery for 1 week, which was comparable to what could only be achieved after leaving an unseeded valve in the same site for 1 month. A small proportion of the cells further differentiates into a smooth muscle cell phenotype, as shown by the approximately 2% cells staining positive for heavy chain myosin in the IP-preseeded valves remaining in the pulmonary position for 1 month.

Stem cells were attracted by the matrix after IP implantation as well as after implantation in the circulation of both unseeded and IP-preseeded valves in the sheep. In the first approach, we used a CD172a antibody labelling macrophages6 and stem cells.9,25 We saw a large presence of these cells in the all samples, confirming the findings of Campbell et al.6 Comparing the fractions of CD172a+ and MHC II+ cells, we see that there is a large difference that is remarkable because macrophages should be MHC II+.26 Two possible explanations are apparent: (i) since the material is non-immunogenic and coated with host proteins, the macrophages are not exerting their function by an MHC II-mediated pathway; or (ii) the CD172a+ fraction is made up of a large proportion of stem/progenitor cells. Therefore, we also studied two markers expressed by stem/progenitor cells. Regarding CD34+ cells, representing 0.5% of the cells in peripheral blood,27 we found already an enrichment in the IP test samples. Despite the fact that some numerical variation was observed, these cells were not differently present in any of the other samples. More interesting were the findings with CD117+ cells.28 During spontaneous seeding, these cells could only be detected after the valves remained in the pulmonary position for 1 month. On the contrary, the IP seeding primed the matrix with a small fraction of these cells, which remained present in the IP-preseeded valves at both time points.

For the identification of endothelial cells, we chose an antibody directed against the ecNOS.29 ecNOS provides additional information regarding the endothelial function,30 but more importantly the commonly used markers, von Willebrand Factor (vWF) or CD31, might yield false positives in this particular setting. Being part of the blood clotting cascade, vWF can insulate the implant surface31 resulting in aspecific staining of the external cell layer. CD31 can also be expressed by T-cells32 and macrophages,33 one of the key cell types in the early foreign body reaction. Scoring the presence of endothelial cells revealed that IP-preseeded valves had a better potential towards re-endothelialization than the nude matrix material. Because of the morphology and patchy development of the endothelial layer in the preseeded valves, we think that the cells are originating from circulating progenitors that home to the newly deposited matrix or are attracted by the deposited cells. In controls, on the other hand, the observed layer of endothelial cells is the external layer of pannus, originating from native vessel cell overgrowth. This is an important finding since it is known that in normal aortic and pulmonary valves the endothelium regulates the anti-thrombogenicity34 and causes an NO-dependent regulation of the intrinsic contractility of the leaflets. Furthermore, myofibroblasts enable this intrinsic contractility by acting on NO, endothelin-1, and prostacyclin released by the endothelium.30

These findings clearly illustrate the potential of these cells to revitalize the stabilized biological matrix with myofibroblasts. Furthermore, the 1 month implants already showed signs of spontaneous re-endothelialization. The process of re-cellularization seems to be self-limiting, since the amount of newly deposited material is not continuously increasing and is getting covered by endothelium. This should prevent new cells from adhering and contributing to the re-cellularization process.

Although we have used a stabilized biological matrix, the cells obtained by peritoneal seeding are able to modify this matrix as well as to deposit their own. An important issue that remains to be studied in this regard is whether or not repopulation is improving the compliance of the material.

This biohybrid valve constructed out of xenogeneic matrix material and autologous cells combines the reliability of the matrix with the viability of the cells. At present, the origin of these cells and the mediators, such as interleukins, integrins, etc. are largely unknown. We know that the cells are of mesenchymal origin and can differentiate into the appropriate phenotype, i.e. myofibroblast, for cell repopulation. Considering the mediators, we also know that the cells and possibly the differentiation are not sustained by a standard cell-culturing medium. At present, the group of Campbell5,6 has shown that the cells are possibly derived from macrophages attracted to the material. The present study provides evidence for our alternative hypothesis that the repopulation, although mediated or initiated by macrophages, is (haematopoietic) stem cell derived.


   Acknowledgements
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
References
 
The project was partially funded by Fonds voor Wetenschappelijk Onderzoek (G.0549.06). The authors wish to thank lab assistants Ruth Plusquin, Veerle Leunens, Monique Vercalsteren, and Kristof Reyniers.

Conflict of interest: none declared.


   Footnotes
 
This paper was guest edited by Prof. Julie H. Campbell, University of Queensland, Australia


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 

  1. Butany J, Fayet C, Ahluwalia MS, Blit P, Ahn C, Munroe C, Israel N, Cusimano RJ, Leask RL. Biological replacement heart valves. Identification and evaluation. Cardiovasc Pathol (2003) 12:119–139.[CrossRef][Web of Science][Medline]
  2. Rabkin E, Schoen FJ. Cardiovascular tissue engineering. Cardiovasc Pathol (2002) 11:305–317.[CrossRef][Web of Science][Medline]
  3. Campbell JH, Efendy JL, Campbell GR. Novel vascular graft grown within recipient's own peritoneal cavity. Circ Res (1999) 85:1173–1178.[Abstract/Free Full Text]
  4. Butler KR, Benghuzzi HA, Puckett A. Morphometric evaluation of tissue-implant reaction associated with ALCAP and TCP bioceramics in vivo. J Invest Surg (2001) 14:139–152.[CrossRef][Web of Science][Medline]
  5. Campbell JH, Efendy JL, Han C, Girjes AA, Campbell GR. Haemopoietic origin of myofibroblasts formed in the peritoneal cavity in response to a foreign body. J Vasc Res (2000) 37:364–371.[CrossRef][Web of Science][Medline]
  6. Bayes-Genis A, Campbell JH, Carlson PJ, Holmes DR Jr, Schwartz RS. Macrophages, myofibroblasts and neointimal hyperplasia after coronary artery injury and repair. Atherosclerosis (2002) 163:89–98.[CrossRef][Web of Science][Medline]
  7. Della Rocca F, Sartore S, Guidolin D, Bertiplaglia B, Gerosa G, Casarotto D, Pauletto P. Cell composition of the human pulmonary valve: a comparative study with the aortic valve—the VESALIO project. Vitalitate Exornatum Succedaneum Aorticum labore Ingegnoso Obtinebitur. Ann Thorac Surg (2000) 70:1594–1600.[Abstract/Free Full Text]
  8. Kuci S, Wessels JT, Buhring HJ, Schilbach K, Schumm M, Seitz G, Loffler J, Bader P, Schlegel PG, Niethammer D, Handgretinger R. Identification of a novel class of human adherent CD34-stem cells that give rise to SCID-repopulating cells. Blood (2003) 101:869–876.[Abstract/Free Full Text]
  9. Vogel W, Grunebach F, Messam CA, Kanz L, Brugger W, Buhring HJ. Heterogeneity among human bone marrow-derived mesenchymal stem cells and neural progenitor cells. Haematologica (2003) 88:126–133.[Abstract/Free Full Text]
  10. Moore MA. PhotoFix: unraveling the mystery. J Long Term Eff Med Implants (2001) 11:185–197.[Medline]
  11. Jansson K, Bengtsson L, Swedenborg J, Haegerstrand A. In vitro endothelialization of bioprosthetic heart valves provides a cell monolayer with proliferative capacities and resistance to pulsatile flow. J Thorac Cardiovasc Surg (2001) 121:108–115.[CrossRef][Web of Science][Medline]
  12. White JK, Agnihotri AK, Latremouille C, Messas E, Carpentier A, Torchiana DF. A method of using the pulmonary trunk to form a trileaflet valve. J Thorac Cardiovasc Surg (2005) 129:677–679.[Free Full Text]
  13. Rabkin E, Hoerstrup SP, Aikawa M, Mayer JE Jr, Schoen FJ. Evolution of cell phenotype and extracellular matrix in tissue-engineered heart valves during in-vitro maturation and in-vivo remodeling. J Heart Valve Dis (2002) 11:308–314.[Web of Science][Medline]
  14. Jackson DG. Human leucocyte heparan sulphate proteoglycans and their roles in inflammation. Biochem Soc Trans (1997) 25:220–224.[Web of Science][Medline]
  15. Deguchi T, Komada Y. Homing-associated cell adhesion molecule (H-CAM/CD44) on human CD34+ hematopoietic progenitor cells. Leuk Lymphoma (2000) 40:25–37.[Web of Science][Medline]
  16. Sunderland CA, McMaster WR, Williams AF. Purification with monoclonal antibody of a predominant leukocyte-common antigen and glycoprotein from rat thymocytes. Eur J Immunol (1979) 9:155–159.[Web of Science][Medline]
  17. Clark BR, Keating A. Biology of bone marrow stroma. Ann N Y Acad Sci (1995) 770:70–78.[Web of Science][Medline]
  18. Ketchedjian A, Jones AL, Krueger P, Robinson E, Crouch K, Wolfinbarger L Jr, Hopkins R. Recellularization of decellularized allograft scaffolds in ovine great vessel reconstructions. Ann Thorac Surg (2005) 79:888–896.[Abstract/Free Full Text]
  19. Brenner RM, Slayden OD, Rodgers WH, Critchley HO, Carroll R, Nie XJ, Mah K. Immunocytochemical assessment of mitotic activity with an antibody to phosphorylated histone H3 in the macaque and human endometrium. Hum Reprod (2003) 18:1185–1193.[Abstract/Free Full Text]
  20. Tang L, Jennings TA, Eaton JW. Mast cells mediate acute inflammatory responses to implanted biomaterials. Proc Natl Acad Sci USA (1998) 95:8841–8846.[Abstract/Free Full Text]
  21. Tang L, Eaton JW. Inflammatory responses to biomaterials. Am J Clin Pathol (1995) 103:466–471.[Web of Science][Medline]
  22. Leferovich JM, Bedelbaeva K, Samulewicz S, Zhang XM, Zwas D, Lankford EB, Heber-Katz E. Heart regeneration in adult MRL mice. Proc Natl Acad Sci USA (2001) 98:9830–9835.[Abstract/Free Full Text]
  23. Askari AT, Unzek S, Popovic ZB, Goldman CK, Forudi F, Kiedrowski M, Rovner A, Ellis SG, Thomas JD, DiCorleto PE, Topol EJ, Penn MS. Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration in ischaemic cardiomyopathy. Lancet (2003) 362:697–703.[CrossRef][Web of Science][Medline]
  24. Yperman J, De Visscher G, Holvoet P, Flameng W. Molecular and functional characterization of ovine cardiac valve-derived interstitial cells in primary isolates and cultures. Tissue Eng (2004) 10:1368–1375.[Web of Science][Medline]
  25. Handgretinger R, Gordon PR, Leimig T, Chen X, Buhring HJ, Niethammer D, Kuci S. Biology and plasticity of CD133+ hematopoietic stem cells. Ann N Y Acad Sci (2003) 996:141–151.[Web of Science][Medline]
  26. Ahn JS, Konno A, Gebe JA, Aruffo A, Hamilton MJ, Park YH, Davis WC. Scavenger receptor cysteine-rich domains 9 and 11 of WC1 are receptors for the WC1 counter receptor. J Leukoc Biol (2002) 72:382–390.[Abstract/Free Full Text]
  27. Krause DS, Fackler MJ, Civin CI, May WS. CD34: structure, biology, and clinical utility. Blood (1996) 87:1–13.[Free Full Text]
  28. Rombouts WJ, Ploemacher RE. Primary murine MSC show highly efficient homing to the bone marrow but lose homing ability following culture. Leukemia (2003) 17:160–170.[CrossRef][Web of Science][Medline]
  29. Laufs U, Endres M, Stagliano N, Amin-Hanjani S, Chui DS, Yang SX, Simoncini T, Yamada M, Rabkin E, Allen PG, Huang PL, Bohm M, Schoen FJ, Moskowitz MA, Liao JK. Neuroprotection mediated by changes in the endothelial actin cytoskeleton. J Clin Invest (2000) 106:15–24.[Web of Science][Medline]
  30. Pompilio G, Rossoni G, Sala A, Polvani GL, Berti F, Dainese L, Porqueddu M, Biglioli P. Endothelial-dependent dynamic and antithrombotic properties of porcine aortic and pulmonary valves. Ann Thorac Surg (1998) 65:986–992.[Abstract/Free Full Text]
  31. Pankowsky DA, Ziats NP, Topham NS, Ratnoff OD, Anderson JM. Morphologic characteristics of adsorbed human plasma proteins on vascular grafts and biomaterials. J Vasc Surg (1990) 11:599–606.[CrossRef][Web of Science][Medline]
  32. Caligiuri G, Rossignol P, Julia P, Groyer E, Mouradian D, Urbain D, Misra N, Ollivier V, Sapoval M, Boutouyrie P, Kaveri SV, Nicoletti A, Lafont A. Reduced immunoregulatory CD31+ T cells in patients with atherosclerotic abdominal aortic aneurysm. Arterioscler Thromb Vasc Biol (2006) 26:618–623.[Abstract/Free Full Text]
  33. Curat CA, Miranville A, Sengenes C, Diehl M, Tonus C, Busse R, Bouloumie A. From blood monocytes to adipose tissue-resident macrophages: induction of diapedesis by human mature adipocytes. Diabetes (2004) 53:1285–1292.[Abstract/Free Full Text]
  34. Ku DD, Nelson JM, Caulfield JB, Winn MJ. Release of endothelium-derived relaxing factors from canine cardiac valves. J Cardiovasc Pharmacol (1990) 16:212–218.[Web of Science][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 CirculationHome page
F. J. Schoen
Evolving Concepts of Cardiac Valve Dynamics: The Continuum of Development, Functional Structure, Pathobiology, and Tissue Engineering
Circulation, October 28, 2008; 118(18): 1864 - 1880.
[Abstract] [Full Text] [PDF]

Home page
 J. Thorac. Cardiovasc. Surg.Home page
G. De Visscher, H. Blockx, B. Meuris, H. Van Oosterwyck, E. Verbeken, M.-C. Herregods, and W. Flameng
Functional and biomechanical evaluation of a completely recellularized stentless pulmonary bioprosthesis in sheep.
J. Thorac. Cardiovasc. Surg., February 1, 2008; 135(2): 395 - 404.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
28/11/1389    most recent
ehl422v1
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (2)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by De Visscher, G.
Right arrow Articles by Flameng, W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by De Visscher, G.
Right arrow Articles by Flameng, W.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?