Pre-operative in vitro fibroblast coating of porous polyethylene compound grafts – Cell survival in vivo and effects on biocompatibility

Abstract

Background:

Key factors for successful porous polyethylene (PPE) implantation are rapid vascularization and low inflammatory response. Dermal fibroblasts produce a variety of pro-angiogenic and immunmodulatory factors.

Objective:

The aim of this tissue engineering study was to investigate whether coating PPE implants with dermal fibroblasts in vitro is sustainable in vivo and whether the kinetics of blood vessel ingrowth and immunological responses are hereby affected.

Methods:

PPE implants were cultured with syngeneic GFP-transfected dermal fibroblasts. Cells on the biomaterial were quantified before implantation into dorsal skinfold chamber preparations of C57Bl/6 mice. Uncoated implants served as controls. Angiogenic activity and leukocyte-endothelial cell interactions were repeatedly analyzed. After 10 days, mechanical integration was measured and surviving fluorescently labeled fibroblasts were quantified. Expression of inflammatory cytokines was assessed by quantitative real time-reverse transcription PCR.

Results:

PPE implants were successfully coated with dermal fibroblasts in vitro and 69% of the cells were still detectable at the end of observation. Angiogenic parameters increased during the observation period in both groups. IL-2, IL17A and IL-10 tended to be increased in coated implants, but did not affect leukocyte-endothelial cell interactions.

Conclusions:

Dermal fibroblast-coating of porous polyethylene implants is feasible and sustainable in vivo. Alone it does not improve biocompatibility but may be beneficial in combination with specific growth factor supplements.

Keywords

Tissue engineering porous polyethylene Medpor dermal fibroblasts angiogenesis

1. Introduction

Tissue engineering using biomaterials in combination with living cells or extracellular components is a promising concept to enhance biocompatibility and function [1]. Biointegration is impaired particularly if an application of a synthetic biomaterial is required at less vascularized sites of the body, for example in reconstructive augmentation surgery or in revision rhinoplasty. The initial period after implantation before the ingrowth of newly formed blood vessels into biomaterial scaffolds is especially prone to infection which can lead to rejection of the implant [2–4]. Hence, tissue engineering approaches aim to support a rapid vascularization and to limit the initial inflammatory host response to the implant. For long term biocompatibility, the composition of the implant material also plays an important role because the toxicity of degradation products may cause an additional inflammatory stimulus [5]. Porous polyethylene (Medpor^®, Porex Surgical Inc., Newnan, GA), which was analyzed in this study is a non-resorbable compound which has been utilized in reconstructive surgery for several decades [6]. Its structure with pore sizes of 100–200 µm promotes the ingrowth of fibrous tissue and it has been routinely applied for the reconstruction of bone defects as well as for other surgical indications, such as otoplasty [7,8]. Despite low reported complication rates [9], investigative approaches have attempted to overcome the aforementioned inherent limitations of implant material biocompatibility. For example, while the properties of porous polyethylene are intriguing for the application in middle face reconstructive procedures, relatively high complication rates have been reported for rhinoplasties where the availability of vital tissue to cover the implant is oftentimes limited [10,11]. Most recently, studies demonstrated that coating porous polyethylene implants with extracellular matrix (ECM) components and vascular endothelial growth factor (VEGF), decreased the initial inflammatory response of the host [12]. Others showed that vitalizing implants with xenogeneic chondrocytes and platelet-rich plasma (PRP) induced an accelerated vascularization in immunodeficient mice [13], while surface coating with components of the plasminogen activation complex improved mechanical integration [14]. Regarding these results and envisioning future clinical applications, we investigated the feasibility and potential benefit of a priori “vitalized” porous polyethylene implants with syngeneic dermal fibroblasts before implantation into immunocompetent mice. Dermal fibroblasts are an easily accessible inexhaustible cell source with favorable immunologic properties and able to continuously produce ECM, as well as a broad spectrum of proangiogenic cytokines [15]. In addition, dermal fibroblasts can function as immunoregulatory cells and are potentially capable of inhibiting local T-cell-activation [16,17]. We analyzed their viability in vivo and their effect on the kinetics of vascularization of the biomaterial as well as the initial inflammatory response after implantation into dorsal skinfold chambers of C57/Bl6 mice. Along with repeated in vivo fluorescence microscopy analysis, the expression of immunoregulatory cytokines as well as matrix metalloproteinases (MMPs)-2 and -9 which are associated with tissue remodeling processes [18], were analyzed in dermal fibroblast-coated compound grafts and compared to uncoated porous polyethylene implants.

2. Materials and methods

2.1. Animals

13 male C57Bl/6 mice (Charles River, Sulzfeld, Germany) served as experimental animals for this study. All experimental procedures performed were in accordance with institutional and governmental guidelines (Regierung von Oberbayern, Munich, Germany). All surgical procedures were performed under anesthesia with ketamine (100 mg/kg Ketavet^®; Parke-Davis, Berlin, Germany) and xylazine (15 mg/kg Rompun^®; Bayer, Leverkusen, Germany).

2.2. Isolation and green fluorescent protein (GFP)-transfection of murine fibroblasts

Dermal fibroblasts were isolated from a single C57Bl/6 mouse. After sacrifice, the dorsal skin was cut into squares of 3 × 3 mm² and all subcutaneous tissue was removed. Incubation for 60 min at 37°C in 0.3% trypsin (Sigma, Deisenhofen, Germany) enabled the removal of the epidermis. Samples were placed into 6-well cell culture plates (Falcon, BD, Franklin Lakes, NJ) under sterile glass covers. Standard growth medium (DMEM; Sigma-Aldrich, Munich, Germany) was added and regularly changed until a confluent cell population was visible and verified light-microscopically as a pure fibroblast culture. For GFP-transfection of dermal fibroblast cultures, 5 µl GFP-plasmid (Plasmid 12091, Addgene, Cambridge, MA) and reduced serum medium (Opti-MEM^®; Invitrogen, Karlsruhe, Germany) were mixed at a ratio of 1:50. 5 µl Lipofectamin (Invitrogen, Karlsruhe, Germany) was combined with 1 ml reduced serum medium and incubated for 5 min. Both solutions were then combined and incubated for 20 min, thereafter 100 µl per well were added to the dermal fibroblast cell cultures. After incubation with media for 72 h, the transfected cells were selected by adding 1 ml of antibiotic per well herby establishing pure cultures of GFP-transfected dermal fibroblasts.

2.3. PEE coating and dermal fibroblast quantification

Porous polyethylene implants (Medpor^®; Porex Surgical Inc., Newnan, GA) were cut into 3 × 3 × 0.25 mm³ samples and fixated in 6-well culture plates. GFP-transfected dermal fibroblasts were added to the wells and cultured for 14 days until a dense layer of fluorescent cells was detectable on the implant material under the fluorescence microscope. To quantify cell density, the implants were analyzed using confocal laser scanning microscopy (Zeiss LSM 510, Goettingen, Germany) before implantation. The images of multiple focal planes were merged into one two dimensional projection to capture all fluorescent cells. The percentage of the implant surface covered with fluorescent cells was determined with the imaging program KS 400 (Carl Zeiss Vision, Hallbergmoos, Germany). This analysis was undertaken before implantation into the dorsal skinfold chamber and repeated after explantation.

2.4. The dorsal skinfold chamber

The procedure has been previously described in detail [19,20]. Under anesthesia, the extended dorsal skin of the mouse was surgically clamped in a double layer between two symmetrical titanium frames after hair removal with depilatory cream (Plica med Creme^®, Asid Bonz GmbH, Bieblingen, Germany). On one side, a circular area of 15 mm diameter consisting of skin, subcutaneous tissue and striated skin muscle was removed. The contralateral muscle was covered with a sterile, removable glass coverslip fitted into a titanium frame. After a 48 h recovery period, the surgical field was microscopically inspected to ensure intact microcirculation and absence of inflammation. The polyethylene sample was placed centrally into the chamber and onto the striated muscle after removal of the coverslip which was then replaced.

2.5. In vivo fluorescence microscopy and microcirculatory analysis

Mice were immobilized in a Perspex tube providing a breathing hole for sufficient air supply, as well as a longitudinal opening through which the dorsal skinfold chambers could protrude. The animals remained conscious throughout the procedure and showed no signs of discomfort. The tube was attached to a specifically designed stage (Effenberger, Munich, Germany) under a modified fluorescence microscope (Axiotech Vario; Zeiss, Goettingen, Germany). FITC-labeled dextran (Sigma, Deisenhofen, Germany; MW 500,000; 0.05–0.1 mL of a 5% solution in 0.9% NaCl) and Rhodamine 6G (Molecular Probes, Eugene, OR; 0.04 mL of a 0.05% solution in 0.9% NaCl) served as markers and were injected into the tail vein. FITC-labeled dextran stains blood plasma and illuminates perfused vessels and microvessels under the fluorescence microscope. Rhodamine 6G is taken up by leukocyte mitochondria, highlighting white blood cells and allowing their quantification as well as an analysis of interactions with the vascular endothelium.

On day 5 after biomaterial implantation, six regions of interest (ROI) per animal were randomly selected, three in the center of the porous polyethylene implant and three in adjacent connective tissue. The same ROIs were sought out again using an x-y-micrometer-stage (Effenberger, Munich, Germany) and analyzed on day 10. In vivo fluorescence microscopic images were acquired by a CD camera (Sony XC-77CE; Sony, Cologne, Germany) and recorded on digital tapes (Sony DVCAM DSV 45P; Sony, Cologne, Germany) for subsequent off-line analysis. Parameters for angiogenic activity. i.e. functional vessel density (fvd), red blood cell velocities (vRBC), and vessel diameters (d) were measured using a specific software (Cap Image; Zeintl, Heidelberg, Germany) as previously described [21]. White blood cells were analyzed regarding the leukocyte flux, which was quantified by counting the number of cells crossing a predefined line in one vessel in 30 s. Rolling and adherent leukocytes were differentiated – rolling was defined as at least 50% of red blood cell velocity in the same vessel, adherent leukocytes were stationary for at least 30 s/mm² of vessel wall surface.

2.6. Measurement of dynamic breaking strength

The force in cN/mm² required to dislocate the implant form the host tissue was measured 10 days after implantation directly after sacrifice as described previously [4,22].

2.7. Molecular biology

After explantation of the implants, quantification of matrix metalloproteinases (MMPs)-2, -9, as well as cytokines IFN-γ, TNF-α, IL-1β, IL-2, IL-4, IL-10 and IL-17A was performed in tissue samples taken from both experimental groups. The analysis was conducted by means of quantitative real time-reverse transcription polymerase chain reaction (qRT-PCR) in a lightcycler system (Lightcycler 3.5; Roche, Penzberg, Germany) using the Fast Start Master PLUS SYBR Green kit (Roche, Penzberg, Germany).

2.8. Experimental protocol

Porous polyethylene implants were coated with syngeneic murine GFP-transfected dermal fibroblasts. The percentage of the implant material surface covered with fluorescent cells was quantified immediately before implantation using confocal microscopy. These compound graft constructs were implanted into dorsal skinfold chambers of male C57/Bl6 mice ( $n = 6$ ). Control animals received uncoated polyethylene implants ( $n = 6$ ). In vivo fluorescence microscopy was performed on days 5 and 10 after implantation. Thereafter, the dynamic breaking strength assessment of implant dislocation was performed immediately post exitum and the remaining fluorescent cells on the explanted porous polyethylene implants were quantified. Subsequently, a real-time RT-PCR analysis was performed on samples taken from implant material, as well as connective tissue from the implant site from both groups to evaluate expression of MMP-2 and -9, as well as IFN-γ, TNF-α, IL-1β, IL-2, IL-4, IL-10, and IL-17A, respectively.

2.9. Statistical analysis

Results are presented as mean ± SEM. Data were evaluated non-parametrically using Mann–Whitney rank sum test and repeated measures analysis on ranks (Sigma Stat; Jandel Corp., San Rafael, CA). P values smaller than 5% were considered significant.

3. Results

3.1. Coating porous polyethylene implants with dermal fibroblasts

A pure dermal fibroblast culture was successfully isolated from the dorsal skin of a C57Bl/6 mouse (Fig. 1A). After GFP-transfection (Fig. 1B), the cells were cultured in vitro on 3 × 3 × 0.25 mm³ porous polyethylene implants. Before implantation, confocal microscopy showed a homogenous distribution of fluorescently labeled dermal fibroblasts on the implants (Fig. 1C). Quantitative analysis of the cell density after merging confocal images of multiple depth planes into one two dimensional maximum projection (Fig. 1D) indicated that on average 20.0% ( $n = 6$ ) of the three-dimensional implant surface was covered with fluorescent cells. The same analysis was undertaken after explantation of the implants. At this point, on average 13.8% of the implant surface remained covered by fluorescent cells, indicating that the majority (69%) of the originally implanted dermal fibroblasts were still vital at the end of the observation period (Fig. 2).

Fig. 1.

Dermal fibroblast culture established from murine dorsal skin before transfection (A). Confocal microscopic image of a GFP-transfected dermal fibroblast culture (B). Single plane confocal microscopic finding of GFP-transfected dermal fibroblasts cultivated on porous polyethylene (Medpor^®) implant material in vitro (C). Multiple confocal microscopic images of different depth planes of one region of the implant merged into a two-dimensional image for cell quantification (D). All scale bars: 200 µm.

Fig. 2.

Quantification of GFP-positive cells on the implants ( $n = 6$ ) with confocal microscopy prior to implantation and after explantation on day 10 showing that 69% of cells remained vital over the period of observation.

3.2. Angiogenesis and microhemodynamics

Porous polyethylene implants were implanted into dorsal skinfold chambers of C57Bl/6 mice (Fig. 3A, B). Quantitative analysis of angiogenic activity was performed twice during the period of observation – i.e. on days 5 and 10 – in the implant material and the adjacent connective tissue by in vivo fluorescence microscopy (Fig. 3C). All angiogenic parameters – i.e. fvd, vRBC, and d – increased during the observation period (Fig. 4). Sufficient blood flow for ingrowing tissue was established in the porous polyethylene implants in both groups between days 5 and 10 after implantation (Fig. 4B). However, there were no significant differences between the study group ( $n = 6$ ) and the control group ( $n = 6$ ) at both time points.

Fig. 3.

Porous polyethylene implant (3 × 3 × 0.25 mm³) implanted onto striated muscle tissue in a dorsal skinfold chamber ((A) and (B)). In vivo fluorescence microscopy for the analysis of angiogenic activity highlights perfused vessels (C). Scale bar: 200 µm.

Fig. 4.

Quantitative microcirculatory analysis. Analysis of regions of interest (ROIs) in the implant material and adjacent connective tissue 5 and 10 days after implantation show no significant differences between mice with fibroblast-coated implants ( $n = 6$ ) and controls ( $n = 6$ ) regarding functional vessel density (A), vessel diameter (C) and red blood cell velocity (D). Figure (A) shows combined blood vessel density values from 6 ROIs per mouse analyzed in the implant material and the adjacent connective tissue, while (B, C and D) show values only from the 3 ROIs within the implants. Here values increased significantly ( $^{*} p < 0.05$ ) between day 5 and day 10 after implantation in both groups indicating that sufficient implant vascularization was achieved at the final observation time point. All values shown ± SEM.

3.3. Inflammatory response

Analysis of the leukocyte-endothelial cell interactions by in vivo fluorescence microscopy showed no significant differences between vitalized ( $n = 6$ ) and non-coated implants ( $n = 6$ ) at both analyzed time points (data not shown).

3.4. Cytokine analysis

The quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) analysis of immunmodulatory cytokines and MMPs did not show any significant expression differences between the study group ( $n = 6$ ) and control group ( $n = 6$ ). The concentrations for IL-2, Il-10 and IL-17 as well as MMP-2 and MMP-9 tended to be increased in dermal fibroblast-coated implants (Fig. 5).

Fig. 5.

Ex vivo RTPCR-analysis measuring the expression of cytokines indicates that MMP-2 and -9, as well as ILl2, IL-10, and IL-17 A tended to be increased in the dermal fibroblast-coated implants ( $n = 6$ ) at the end of the observation period compared to uncoated implants ( $n = 6$ ). Differences were not statistically significant.

3.5. Mechanical integration

Dermal fibroblast-coated, as well as uncoated porous polyethylene implants were well integrated into the host tissue 10 days after implantation. The force necessary to dynamically dislocate the implants out of the implant bed did not differ (Fig. 6).

Fig. 6.

The force necessary to dislocate the implant from the host tissue did not differ between coated implants ( $n = 6$ ) and uncoated controls ( $n = 6$ ) 10 days after implantation.

4. Discussion

We investigated the feasibility of coating porous polyethylene implants with dermal fibroblasts and analyzed their effect on the vascular integration process and the initial inflammatory response of the host organism, two key parameter for successful biomaterial integration [23]. The scaffolds were implanted into dorsal skinfold chambers. This particular model is well established for repeated analysis of microcirculatory and inflammatory parameters after tissue transplantation and biomaterial implantation by in vivo fluorescence microscopy. The range of reported applications includes the analysis of tumor angiogenesis [24,25], exocrine function of parathyroid tissue [26] and vascularization of ovarian follicles [27]. For biomaterial compatibility studies, the possibility to repetitively analyze tissue integration parameters in an adult, immunocompetent host organism is unmatched by alternate models like the chorion allantois membrane (CAM) assay [23,28].

4.1. Survival of the dermal fibroblast-coating on porous polyethylene implants in vivo

Sustainably seeding cells onto a hydrophobic scaffold prior to implantation is a challenge. Varying strategies, such as application of cells in a hydrogel [29], or soaking implants in a cell suspension have been reported [30]. Others suspended chondrocytes in platelet rich plasma (PRP), and applied the viscous solution to porous polyethylene [13]. We cultivated the biomaterial with dermal fibroblasts in vitro prior to implantation to ensure that cells were sufficiently adherent to the material. GFP-expression is a well-established indicator of gene expression and cell viability [31], and GFP-transfection of the dermal fibroblasts enabled the quantification of coverage by vital cells before implantation and again after explantation. The unique surface structure of porous polyethylene implants determines the cell density attainable by in vitro cultivation and our results correspond to cell densities reported for similar materials [32]. Confocal fluorescence microscopic analysis of the implants after explantation confirmed that the GFP-transfected dermal fibroblasts had remained attached to the porous polyethylene in vivo and indicated that on average, 69% of the implanted fibroblasts remained vital in the host organism for the period of observation. While it has been reported that dermal fibroblasts show robust survival in vivo over several weeks when seeded onto ligament-like collagen scaffolds [33], this finding indicates that three dimensional, porous biomaterial scaffolds can be successfully ‘vitalized’ with dermal fibroblasts in vitro and that cell survival is obtainable in vivo without additional growth factor or nutrient application.

4.2. Immune response and microcirculation

Besides being easily accessible and rapidly expandable in vitro, dermal fibroblasts have been shown to possess a low rejection and sensitization potential in the host organism and are routinely applied in commercially available therapeutic products, such as skin substitutes for ulcer treatment [34]. For future clinical application, dermal fibroblasts could potentially be harvested directly from the patient pre-operatively and expanded in vitro prior to the procedure. Hereby, adverse immune responses to the cells could be avoided completely. To mirror this intriguing scenario, in this study, we used syngeneic dermal fibroblasts isolated from a genetically identical donor animal. Differences in immune response between the two groups were therefore assumed to be attributable to fibroblast activity in vivo, rather than the host response to the cells themselves. Interestingly, fibroblasts can exhibit certain immunosuppressive properties by influencing IFN-γ production by T-cells [16]. However, our analysis of leukocyte-endothelial cell interactions in the implant material and the surrounding connective tissue as well as the assessment of a broad spectrum of immunomodulatory cytokines did not show any significant differences between fibroblast-coated and untreated implants. While these findings indicate that dermal fibroblast-coating does not reduce the initial inflammatory response of the host organism to porous polyethylene, these data also confirm, that syngeneic dermal fibroblasts retain a low immunogeneic potential after in vitro expansion in an immunocompetent host. Previous studies showed that the transient initial inflammatory response to porous polyethylene implants could be reduced by the local application of high doses of VEGF and ECM components [12]. While dermal fibroblasts are potent producers of ECM components and VEGF [35], extensive concentrations of stimulating factors are not induced by cell implantation alone. Their supplementation may therefore be a strategy to influence long term biocompatibility, particularly of biomaterials with less favorable biocompatibility profiles than porous polyethylene [36].

The analysis of immunoregulatory cytokines as well as MMPs-2 and -9 in the biomaterial after explantation showed that the expression of certain cytokines considered to promote inflammation – i.e. IL-2 and IL-17, as well the anti-inflammatory cytokine IL-10 and MMP-2 and -9 tended to be increased in the coated implants. Potentially, this can be interpreted as the sequelae of an unspecific T-cell activation and upregulation of ECM synthesis induced by dermal fibroblasts in the host organism. Increased MMP expression has been associated with angiogenic activity [18], which might be expected in the fibroblast-coated implants. Dermal fibroblasts produce proangiogenic factors; besides VEGF, hepatocyte growth factor (HGF) and angiopoietin-1 (Ang-1) stimulate vascular endothelial cells [37,38]. However, the analysis of angiogenic parameters in the implants and the adjacent connective tissue did not reveal any significant differences between coated and uncoated implants. Ehrmantraut et al. reported an acceleration of vessel ingrowth into porous polyethylene implants by coating with chondrocytes and PRP [13]. Coating with PRP alone did not influence the kinetics of vascularization, endorsing the use of cultivated living cells on biomaterials in order to sustainably support the integration process. In view of their results and our own findings, it can be hypothesized that an additional initial application of nutrients or growth factors might be necessary to ensure prolonged viability and productivity of the implanted vitalizing cells.

4.3. Mechanical integration

Functional breaking strength, as measured in this study, is a highly relevant parameter for functional implant integration. Our results showed no statistically significant differences in breaking strength required for implant removal between the two groups 10 days after implantation. This is in line with our prior experiments where breaking strength was measured at a similar time point [12]. Here too, breaking strength did not differ, despite improved microvascular integration in the experimental group at the respective time point. Other recent studies analyzing biomechanical properties of porous polyethylene implants in a mouse model suggest that functionally relevant differences may potentially be observed at later time points, around 6 weeks after biomaterial implantation [39]. Therefore, a longer observation period may be required to reliably assess differences in functional integration based on our experimental approach. Remarkably, one recent study did show a difference in mechanical integration at an early time point, i.e. 14 days after implantation. Here, tissue plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), and plasminogen activator inhibitor-1 (PAI-1)-deficient mice were compared to control mice [14]. In deficient mice, collagen-formation was measurably impaired and mechanical integration was worse. These findings suggest that a severe intervention may influence the early integration of this well-established biomaterial. An additional high-dose application of nutrients or growth factors in combination with fibroblasts, as suggested above, could potentially provide a significant stimulus, however, this needs to be evaluated in further experiments.

In conclusion, we could show in this proof of concept study that coating porous polyethylene implants with dermal fibroblasts in vitro is possible and that the majority of implanted cells survive for up to 10 days in vivo. However, dermal fibroblast-coating did not affect the immune response to the implant in an immunocompetent mouse model system or the kinetics of microvascular integration of the biomaterial. For future studies, a combination of dermal fibroblasts with supplemental specific cytokines might sustainably improve biocompatibility of porous polyethylene compound grafts in vivo.

Footnotes

Acknowledgements

This study was supported by Friedrich-Baur-Stiftung (Munich, Germany), Porex Surgical Inc. (Newnan, GA) and the BiomaTICS (Biomaterials, Tissues and Cells in Science) Consortium at the University Medical Center Mainz (Germany).

Conflict of interest

None of the authors has a financial interest in any of the products, devices, or drugs mentioned in this manuscript.

References

Langer

and Vacanti

J.P.

, Tissue engineering, Science 260(5110) (1993), 920–926. doi:10.1126/science.8493529.

Peled

Z.M.

Warren

A.G.

Johnston

and Yaremchuk

M.J.

, The use of alloplastic materials in rhinoplasty surgery: A meta-analysis, Plast. Reconstr. Surg. 121(3) (2008), 85e–95e. doi:10.1097/01.prs.0000299386.73127.a7.

Sclafani

A.P.

Thomas

J.R.

Cox

A.J.

and Cooper

M.H.

, Clinical and histologic response of subcutaneous expanded polytetrafluoroethylene (Gore-Tex) and porous high-density polyethylene (Medpor) implants to acute and early infection, Arch. Otolaryngol. Head Neck Surg. 123(3) (1997), 328–336. doi:10.1001/archotol.1997.01900030110014.

Laschke

M.W.

Haufel

J.M.

Thorlacius

and Menger

M.D.

, New experimental approach to study host tissue response to surgical mesh materials in vivo , J. Biomed. Mater. Res. A 74(4) (2005), 696–704. doi:10.1002/jbm.a.30371.

Griffith

L.G.

, Emerging design principles in biomaterials and scaffolds for tissue engineering, Ann. NY Acad. Sci. 961 (2002), 83–95. doi:10.1111/j.1749-6632.2002.tb03056.x.

Berghaus

, Porous polyethylene in reconstructive head and neck surgery, Arch. Otolaryngol. 111(3) (1985), 154–160. doi:10.1001/archotol.1985.00800050048005.

Berghaus

Stelter

Naumann

and Hempel

J.M.

, Ear reconstruction with porous polyethylene implants, Adv. Otorhinolaryngol. 68 (2010), 53–64.

Cho

Y.R.

and Gosain

A.K.

, Biomaterials in craniofacial reconstruction, Clin. Plast. Surg. 31(3) (2004), 377–385. doi:10.1016/j.cps.2004.03.001.

Yaremchuk

M.J.

, Facial skeletal reconstruction using porous polyethylene implants, Plast. Reconstr. Surg. 111(6) (2003), 1818–1827. doi:10.1097/01.PRS.0000056866.80665.7A.

10.

Stelter

Strieth

and Berghaus

, Porous polyethylene implants in revision rhinoplasty: Chances and risks, Rhinology 45(4) (2007), 325–331.

11.

Berghaus

and Stelter

, Alloplastic materials in rhinoplasty, Curr. Opin. Otolaryngol. Head Neck Surg. 14(4) (2006), 270–277. doi:10.1097/01.moo.0000233599.14671.4a.

12.

Strieth

Weger

Bartesch

Bittmann

Stelter

and Berghaus

, Biocompatibility of porous polyethylene implants tissue-engineered by extracellular matrix and VEGF, Journal of Biomedical Materials Research Part A 93(4) (2010), 1566–1573.

13.

Ehrmantraut

Naumann

Willnecker

Akinyemi

Korbel

Scheuer

et al., Vitalization of porous polyethylene (Medpor^®) with chondrocytes promotes early implant vascularization and incorporation into the host tissue, Tissue Eng. Part A 18(15–16) (2012), 1562–1572. doi:10.1089/ten.tea.2011.0340.

14.

Reichel

C.A.

Hessenauer

M.E.

Pflieger

Rehberg

Kanse

S.M.

Zahler

et al., Components of the plasminogen activation system promote engraftment of porous polyethylene biomaterial via common and distinct effects, PloS One 10(2) (2015), e0116883. doi:10.1371/journal.pone.0116883.

15.

Rhodes

N.P.

Bayon

Chen

Brans

Benne

et al., The use of flow perfusion culture and subcutaneous implantation with fibroblast-seeded PLLA-collagen 3D scaffolds for abdominal wall repair, Biomaterials 31(15) (2010), 4330–4340. doi:10.1016/j.biomaterials.2010.02.010.

16.

Shimabukuro

Murakami

and Okada

, Interferon-gamma-dependent immunosuppressive effects of human gingival fibroblasts, Immunology 76(2) (1992), 344–347.

17.

Haniffa

M.A.

Wang

X.N.

Holtick

Rae

Isaacs

J.D.

Dickinson

A.M.

et al., Adult human fibroblasts are potent immunoregulatory cells and functionally equivalent to mesenchymal stem cells, J. Immunol. 179(3) (2007), 1595–1604. doi:10.4049/jimmunol.179.3.1595.

18.

Page-McCaw

Ewald

A.J.

and Werb

, Matrix metalloproteinases and the regulation of tissue remodelling, Nat. Rev. Mol. Cell Biol. 8(3) (2007), 221–233. doi:10.1038/nrm2125.

19.

Endrich

Asaishi

Gotz

and Messmer

, Technical report – A new chamber technique for microvascular studies in unanesthetized hamsters, Research in Experimental Medicine (Zeitschrift fur die gesamte experimentelle Medizin einschliesslich experimenteller Chirurgie) 177(2) (1980), 125–134.

20.

Menger

M.D.

Laschke

M.W.

and Vollmar

, Viewing the microcirculation through the window: Some twenty years experience with the hamster dorsal skinfold chamber, European Surgical Research (Europaische chirurgische Forschung Recherches chirurgicales europeennes) 34(1–2) (2002), 83–91. doi:10.1159/000048893.

21.

Klyscz

Junger

Jung

and Zeintl

, Cap image – A new kind of computer-assisted video image analysis system for dynamic capillary microscopy, Biomed. Tech. (Berl.) 42(6) (1997), 168–175. doi:10.1515/bmte.1997.42.6.168.

22.

Menger

M.D.

Hammersen

and Messmer

, In vivo assessment of neovascularization and incorporation of prosthetic vascular biografts, Thorac. Cardiovasc. Surg. 40(1) (1992), 19–25. doi:10.1055/s-2007-1020105.

23.

Laschke

M.W.

Harder

Amon

Martin

Farhadi

Ring

et al., Angiogenesis in tissue engineering: Breathing life into constructed tissue substitutes, Tissue Eng. 12(8) (2006), 2093–2104. doi:10.1089/ten.2006.12.2093.

24.

Eichhorn

M.E.

Luedemann

Strieth

Papyan

Ruhstorfer

Haas

et al., Cationic lipid complexed camptothecin (EndoTAG-2) improves antitumoral efficacy by tumor vascular targeting, Cancer Biol. Ther. 6(6) (2007), 920–929. doi:10.4161/cbt.6.6.4207.

25.

Leunig

Yuan

Menger

M.D.

Boucher

Goetz

A.E.

Messmer

et al., Angiogenesis, microvascular architecture, microhemodynamics, and interstitial fluid pressure during early growth of human adenocarcinoma LS174T in SCID mice, Cancer Res. 52(23) (1992), 6553–6560.

26.

Strieth

von Johnston

Eichhorn

M.E.

Enders

Krasnici

Thein

et al., A new animal model to assess angiogenesis and endocrine function of parathyroid heterografts in vivo , Transplantation 79(4) (2005), 392–400. doi:10.1097/01.TP.0000151633.92173.75.

27.

Vollmar

Laschke

M.W.

Rohan

Koenig

and Menger

M.D.

, In vivo imaging of physiological angiogenesis from immature to preovulatory ovarian follicles, Am. J. Pathol. 159(5) (2001), 1661–1670. doi:10.1016/S0002-9440(10)63013-1.

28.

Ribatti

Nico

Vacca

Roncali

Burri

P.H.

and Djonov

, Chorioallantoic membrane capillary bed: A useful target for studying angiogenesis and anti-angiogenesis in vivo , Anat. Rec. 264(4) (2001), 317–324. doi:10.1002/ar.10021.

29.

Monroy

Kojima

Ghanem

M.A.

Paz

A.C.

Kamil

Vacanti

C.A.

et al., Tissue engineered cartilage “bioshell” protective layer for subcutaneous implants, Int. J. Pediatr. Otorhinolaryngol. 71(4) (2007), 547–552. doi:10.1016/j.ijporl.2006.11.025.

30.

Han

S.K.

Chun

K.W.

Gye

M.S.

and Kim

W.K.

, The effect of human bone marrow stromal cells and dermal fibroblasts on angiogenesis, Plast. Reconstr. Surg. 117(3) (2006), 829–835. doi:10.1097/01.prs.0000201458.80364.31.

31.

Chalfie

Euskirchen

Ward

W.W.

and Prasher

D.C.

, Green fluorescent protein as a marker for gene expression, Science 263(5148) (1994), 802–805. doi:10.1126/science.8303295.

32.

Edwards

S.L.

Church

J.S.

Alexander

D.L.

Russell

S.J.

Ingham

Ramshaw

J.A.

et al., Modeling tissue growth within nonwoven scaffolds pores, Tissue Eng. Part C Methods 17(2) (2011), 123–130. doi:10.1089/ten.tec.2010.0182.

33.

Bellincampi

L.D.

Closkey

R.F.

Prasad

Zawadsky

J.P.

and Dunn

M.G.

, Viability of fibroblast-seeded ligament analogs after autogenous implantation, J. Orthop. Res. 16(4) (1998), 414–420. doi:10.1002/jor.1100160404.

34.

Falanga

Margolis

Alvarez

Auletta

Maggiacomo

Altman

et al., Rapid healing of venous ulcers and lack of clinical rejection with an allogeneic cultured human skin equivalent. Human Skin Equivalent Investigators Group, Arch. Dermatol. 134(3) (1998), 293–300.

35.

Trompezinski

Berthier-Vergnes

Denis

Schmitt

and Viac

, Comparative expression of vascular endothelial growth factor family members, VEGF-B, -C and -D, by normal human keratinocytes and fibroblasts, Exp. Dermatol. 13(2) (2004), 98–105. doi:10.1111/j.0906-6705.2004.00137.x.

36.

Rucker

Laschke

M.W.

Junker

Carvalho

Schramm

Mulhaupt

et al., Angiogenic and inflammatory response to biodegradable scaffolds in dorsal skinfold chambers of mice, Biomaterials 27(29) (2006), 5027–5038. doi:10.1016/j.biomaterials.2006.05.033.

37.

Martin

T.A.

Harding

K.G.

and Jiang

W.G.

, Regulation of angiogenesis and endothelial cell motility by matrix-bound fibroblasts, Angiogenesis 3(1) (1999), 69–76. doi:10.1023/A:1009004212357.

38.

Hurley

J.R.

Balaji

and Narmoneva

D.A.

, Complex temporal regulation of capillary morphogenesis by fibroblasts, Am. J. Physiol. Cell Physiol. 299(2) (2010), C444–C453. doi:10.1152/ajpcell.00572.2009.

39.

O’Sullivan

N.A.

Kobayashi

Ranka

M.P.

Zaleski

K.L.

Yaremchuk

M.J.

Bonassar

L.J.

et al., Adhesion and integration of tissue engineered cartilage to porous polyethylene for composite ear reconstruction, Journal of Biomedical Materials Research Part B, Applied Biomaterials 103(5) (2015), 983–991. doi:10.1002/jbm.b.33269.