Conceived and designed the experiments: CFB BMW KM LAM. Performed the experiments: CFB BMW SCR. Analyzed the data: CFB BMW. Contributed reagents/materials/analysis tools: CFB LAM. Wrote the paper: CFB BMW LAM.
The authors have declared that no competing interests exist.
The use of biologic mesh to repair abdominal wall defects in contaminated surgical fields is becoming the standard of practice. However, failure rates and infections of these materials persist clinically. The purpose of this study was to determine the mechanical properties of biologic mesh in response to a bacterial encounter.
A rat model of
The overall rate of staphylococcal mesh colonization was 81% and was comparable in the ADM and SIS groups. Initially (day 0) both biologic meshes had similar biomechanical properties. However after implantation, the SIS control material was significantly weaker than ADM at 20 days (
The biomechanical properties of biologic mesh significantly decline after colonization with MRSA. Surgeons selecting a repair material should be aware of its biomechanical fate relative to other biologic materials when placed in a contaminated environment.
Incisional hernias are one of the most common complications following abdominal surgery. The use of implantable synthetic mesh material has proven to be the preferred method of hernia repair to decrease the recurrence rates
To avoid the potential sequelae of synthetic prosthetic mesh, biological prosthetics have been developed and used for hernia repair. These materials are all essentially composed of an extracellular matrix (ECM) stripped of its cellular components, but differ substantially in their source (porcine small intestine submucosa, porcine dermis or cadaveric human dermis), de-cellularization and sterilization methods
The ability of these materials to resist the influence of bacterial persistence on the implantation site is most likely a function of the bacteria communities, the composition of the biologic mesh and the morphologic properties of its surfaces, as well as the interaction with the host's local tissue defenses. Some researchers believe that an insidious perpetual fight between invading pathogens and the patient's immune system turns the surgical site to an inflamed battleground, resulting in a constant release of inflammatory mediators which subsequently end in mesh degradation and significant loss of function and finally recurrence of the abdominal wall hernia. It is possible that no biological mesh could hope to withstand an overwhelming infection. However, to date, no investigators have addressed the effect of a bacterial colonization on the biomechanical properties of these biologic meshes
This study was approved and monitored by Tulane University's Institutional Animal Care and Use Committee, and all animals were cared for in accordance with guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care International (#4026R).
The bacterial strain, methicillin-resistant
Male Sprague Dawley rats (Charles River Laboratories International, Inc., Wilmington, MA), each ranging from 300 to 500 grams had dorsal subcutaneous pockets created using an established model, described briefly below. The animals were randomly assigned to receive one of the two FDA- approved biologic meshes, for hernia repair, commonly used in clinical practice acellular dermal matrix (ADM; AlloDerm; Life Cell, Branchburg, NJ) or multi-laminate (8-layer) porcine small intestinal submucosa (SIS; Surgsis Biodesign; Cook Biotech, Bloomington, IN). Rectangular implants (2.5×1.5 cm) were fashioned from each material using a sterile plastic template, and rehydrated in sterile saline immediately before implantation. The control (non-infected) animals (
All animals were anesthetized using inhaled isoflurane initially via induction chamber and maintained by a nosecone (1–5% oxygen). The back of each rat was then clipped and cleaned with povidone and allowed to dry for 2 minutes. According to the model by Darouiche and colleagues, the biologic mesh implants were placed under dorsal skin flaps
At ten and twenty days postoperatively, the animals were anesthetized, and a careful dissection was performed to open the dorsal flap. The underlying implant was evaluated carefully and excised, if needed, with surrounding tissue using sterile instruments. One implant per animal was placed in a Petri dish containing 2 ml of 0.9% saline to remain hydrated prior to biomechanical analysis. The other implant was cutinto two equal pieces. One piece was fixed for 24 h in 10% buffered formalin (Fisher Scientific, Fair Lawn, NJ) and processed according to conventional procedures for histologic assessment and the other piece was placed in a tube containing 1 ml sterile saline and immediately analyzed by serial dilution plating for any bacteria present.
A cardiac puncture was then taken after which the animal was humanely euthanized. This blood drawn at study termination was used to determine if there was any hematogenous dissemination of the bacteria which could lead to multi-device colony counts in the same animal. A 100 µl aliquot of whole blood was inoculated on a blood agar plate. Bacterial growth was assessed after the plates were incubated at 37°C for forty-eight hours.
Several initial
For the
The biomechanical properties of each type of biologic mesh were determined by ultimate tensile strength, and modulus of elasticity before and after inoculation with MRSA. These properties are expressed by most biological tissues when a load is applied. These properties were determined immediately after graft explantation. Ultimate tensile strength (i.e. stress) was defined as the maximum force per cross sectional area that is applied to a material. Modulus of elasticity (an indicator of stiffness) was defined as the stiffness or ability to resist deformation (i.e. the tendency of a material to undergo elastic deformation when a force is applied; increase modulus of elasticity = increase stiffness of the material). It is equal to stress versus strain ratio, so an increase in deformation would lead to a decrease in the modulus if the stress were held constant.
The mechanical properties were measured using an electromechanical testing system (MTS Systems Corporation, Eden Prairie, MN) equipped with the ReNew upgrade package 1122 (MTS Systems Corporation, Eden Prairie, MN) and an Instron 1,000 lb load cell (Instron, Norwood, MA). System control and data analysis was accomplished at a sampling rate of 60 Hz with TestWorks 4 software (MTS Systems Corporation, Eden Prairie, MN) in displacement control mode. For each sample, uniaxial strain was applied at a rate of 30 mm/min until failure was detected. Failure was defined as a reduction in applied load of eighty percent of the maximum load. During elongation, force/displacement data was collected and the ultimate tensile strength (peak stress; MPa), strain at break (mm/mm) and Modulus of elasticity (MPa) was recorded for each sample. Once failure was detected, as described above, the test was concluded.
Six sections, five micrometers thick, were cut from each sample, stained with Hematoxylin and Eosin (H&E), and examined using light microscopy. Each slide was assessed and subjectively graded by a pathologist blinded to the treatment group for the following characteristics: inflammation, depth of inflammatory response, neovascularization and cellular re-population response. Histological grading was performed as shown in
Host Response | Score | |
Inflammation | 0–4 PMNs/HPF | 1 |
5–20 PMNs/HPF | 2 | |
>20 PMNs/HPF | 3 | |
Diffuse band-like infiltrate (diffuse to numerous to count) | 4 | |
Depth of Inflammation | Inflammatory cells not present | 1 |
Inflammatory cells present within one-third of tissue matrix | 2 | |
Inflammatory cells present within two-third of tissue matrix | 3 | |
Inflammatory cells present within entire tissue matrix (full-thickness) | 4 | |
Neo-Vascularization | No or rare capillaries | 1 |
Few capillaries (<5 capillaries/HPF) | 2 | |
Many capillaries (5–10 capillaries/HPF) | 3 | |
Abundant capillaries present (granulation tissue) | 4 | |
Cellular re-population | Tissue matrix containing no nuclei of fibroblasts | 1 |
Tissue matrix containing nuclei of fibroblasts within one-third of matrix | 2 | |
Tissue matrix containing nuclei of fibroblasts within two-third of matrix | 3 | |
Tissue matrix containing nuclei of fibroblasts within full thickness of matrix | 4 |
PMNs, polymorphonuclear cells; HPF, high power field;
*Cellular re-population of the acellular collagenous matrix by cellular collagen containing nuclei of fibroblast.
Note. 40 x magnifications.
Data represent the mean ± SEM. Data was analyzed using GraphPad InStat (ver. 3.0; Oberlin Drive, San Diego, CA USA). For continuous variables, three or more group comparisons were analyzed using a one-way analysis of variance with Bonferroni post-test as indicated in the text. Comparison of two groups were done using Mann-Whitney test. For categorical values, Fisher's exact tests were used, and a
All animals had a normal post-operative recovery and none died during the study period. No animals exhibited any drainage from or dehiscence of the surgical wounds or mesh extrusion from the skin. Upon opening the pocket, all implanted materials could be located and separated from its surrounding tissue with minimal dissection. We did not observe any abscesses containing significant amounts of white, pus-like material within the implant pocket or surrounding the implant on gross observation. Some erythema of the pocket tissue was present in the experimental groups but not in control (non-infected) animals. In only 1 of the SIS implants (109 MRSA; 20 days) it was noted to be delaminated and tore upon removal. This animal was excluded from the experimental results. A small fluid collection in the implant area, consistent with seroma/hematoma, was noted in six animals (ADM 3, SIS 3). The control implants had minimal surrounding tissue adhered to their surface. This tissue could simply be peeled away from the implants.
A comparison of the rates of mesh colonization is presented in
Study Group | ADM | SIS | ||
Inoculum size (MRSA) | Day post inoculation | No. Colonized/total (%) | No. Colonized/total (%) | p value |
105 | 10 | 3/6 (50) | 5/6 (83) | 0.54 |
105 | 20 | 4/6 (67) | 6/6 (100) | 0.45 |
109 | 10 | 5/6 (83) | 6/6 (100) | 1.0 |
109 | 20 | 5/6 (83) | 5/6 (83) | 1.0 |
The quantities of
A representative stress-strain curve for the bioprosthesis is shown in
The dashed line indicates the linear region of the curve, the slope of which is the modulus of elasticity. The point labeled M is the proportional limit that corresponds to the end of the linear region of the curve and correlates with the transition from elastic deformation to plastic deformation. During elastic deformation, if the load were removed the material would return to its original size and with no permanent deformation. Once the material progresses to plastic deformation it has undergone permanent deformation and removing the load will no longer return the material to its original size. The point labeled Y is the ultimate tensile strength, which indicates the maximum load applied to the material.
There was no significant difference in the ultimate tensile strength between the un-implanted (day 0) ADM (23.7±1.6 MPa) and SIS (25.9±2.8 MPa). Following implantation, the control mesh experienced a significant time dependent decrease in ultimate tensile strength (
White bars (SIS) and Black bars (ADM) represent the control (non-inoculated) values for the 2 biologic meshes at the different time points. Both materials exhibited the greatest reduction in ultimate strength at 20 days post inoculation with 109 MRSA. * Indicates a statistically significant difference between the control groups. ** Indicates a statistically significant difference between inoculated and control groups. ANOVA
In the bacterial contamination groups, exposure to MRSA appeared to further weaken the materials compared to controls. In addition, significant material differences were observed in response to this bacterial encounter. As shown in
Significant differences in material properties also emerged when colonized ADM was compared with colonized SIS at both 10 and 20 days. Indeed, the following generalizations were noted regarding the mechanical performance of the materials tested in response to 105 cfu MRSA: ultimate strength at 10 days ADM> SIS(p = 0.002), at 20 days ADM>SIS (p = 0.008), in response to 109 cfu MRSA: ultimate strength at 10 days ADM> SIS (p = 0.005), at 20 days ADM = SIS (p = 0.15).
The modulus of elasticity of the un-implanted (day 0) ADM (244.7±22.7 MPa) was slightly lower than SIS (344.2±38.3 MPa) however this was not statistically different (p>0.05). After implantation, control mesh experienced a decrease in the modulus of elasticity (
White bars (SIS) and Black bars (ADM) represent the control (non-inoculated) values for the 2 biologic meshes at the different time points. SIS showed the earliest changes in the modulus of elasticity. * Indicates a statistically significant difference between the control groups. ** Indicates a statistically significant difference between inoculated and control groups. ANOVA.
In the bacterial contamination groups, after inoculation with MRSA (105), the modulus of elasticity did not significantly change compared to control values at 10 days for the ADM or SIS. However as shown in
When colonized ADM was compared with colonized SIS the following generalizations were observed regarding the modulus of elasticity of the materials in response to 105 cfu MRSA: 10 days ADM> SIS (p = 0.002), at 20 days ADM>SIS (p = 0.004); in response to 109 cfu MRSA: modulus of elasticity at 10 days ADM> SIS (p = 0.002), at 20 days ADM = SIS (p = 0.24).
The results of the histological analysis of the meshes are presented in
ADM | ||||||
10 Day | 20 Day | |||||
Control | MRSA 105 | MRSA 109 | Control | MRSA 105 | MRSA 109 | |
Inflammation | 2 | 1 | 3 | 1.7 | 1.3 | 3.2 |
Depth of Inflammation | 2 | 2 | 3 | 3 | 3.5 | 3.4 |
Neo-vascularization | 2 | 2 | 3.2 |
3 | 2.5 | 3.4 |
Cellular re-population | 2 | 3 | 3 | 2.7 | 2.8 | 2.6 |
SIS | ||||||
10 Day | 20 Day | |||||
Control | MRSA 105 | MRSA 109 | Control | MRSA 105 | MRSA 109 | |
Inflammation | 3.5 | 4 | 4 | 1.8 | 3 |
3.5 |
Depth of Inflammation | 2.3 | 2.5 | 2.5 | 2.2 | 2 | 2.8 |
Neo-vascularization | 1 | 1.3 | 1.5 | 2.2 | 1.2 |
2.2 |
Cellular re-population | 1 | 1.3 | 1 | 2.3 | 1.4 |
2 |
*p value ≤0.05 vs. control.
After inoculation with MRSA, the degree and depth of polymorphonucleocyte (PMN) infiltration increased indicating a prominent inflammatory response. This inflammatory response however was related to the inoculum size and mesh material. For example as shown in
All implants ultimately induced neo-vascularization. However compared to SIS, newly formed vessels were easily seen within the ADM after just 10 days. In fact, by day 10 the number of new vessels markedly increased in the ADM mesh exposed to an inoculum of 109 MRSA compared to controls (
A contaminated or infected surgical site is considered a relative contraindication for the use of synthetic mesh material employed to repair abdominal wall defects. As a result, many abdominal wall defects are routinely being repaired with biologic prosthetics. Biologic meshes provide a collagen-rich scaffold that allows cellular in-growth and tissue remodeling, thereby setting the stage for an intact hernia repair
Colonization or adherence of bacteria on the surfaces of a mesh is a prerequisite for mesh-related infection. In our study
First of all, our data confirm the work by others, which reported that biologic meshes are susceptible to bacterial colonization/infection
Beside infection, inflammation has the potential to progressively destroy the structural integrity of these biologic materials
Any implanted biologic mesh most likely relies on vascular in-growth before it acquires any antimicrobial defense to bacteria. Indeed, if neovascularization is inhibited it may significantly hamper both the immune response to the infection as well as the efficacy of intravenous antibiotic use. In our study we observed visible vascular growth as early as 10 days post implantation. These results were similar with that of other investigators
The biomechanical properties (i.e. strength and stiffness) of any material used to repair abdominal wall defects are important in maintaining the structural integrity of the repair. After implantation both biologic meshes lost a significant amount of strength in the absence of bacterial encounter (controls). In fact, a 71% decrease in the strength of control SIS was observed during the first 10 days after implantation in our rat model. By comparison, at this time point, ADM showed a 49% reduction in strength. Other investigators have reported similar findings in animal models early after implantation
As hypothesized, the performance profile of the biologic mesh varied in response to a bacterial encounter. In the group that received ADM, the ultimate tensile strength, was markedly higher than those in the SIS group. Whereas ADM requires both a higher dose, and a longer time period before showing any signs of significant degradation, SIS begins to exhibit signs of degradation sooner than ADM and with much lower doses. Not only did we observe a decrease in material strength in response to a bacterial encounter but also its modulus of elasticity. The decrease in modulus of elasticity was due to an increase in the strain and a decrease in the stress. Both SIS and ADM exhibited a marked decreased modulus of elasticity when inoculated with high dose MRSA (109) after 20 days. This indicates that in addition to a reduction in overall strength, the materials are exhibiting an increased deformation prior to failure. This increase in deformation could be a mechanism of failure for the materials without the ultimate strength needing to be reduced to physiological levels. An increase in the deformation of the material could lead to recurrent hernia formation without the material failing. These results are in line with the scattered data from clinical reports of bulging after implantation in humans
It has been reported that the mean intra-abdominal pressure while standing is 2.7 kPa and 14.3 kPa when coughing
The findings of the present study indicate that our
Infection or colonization of any implant is difficult if not almost impossible to overcome and represent a formidable clinical challenge. The following experimental study highlights some of the concerns with biologic mesh when placed in an infected field. Specifically, we urge caution when considering biologic mesh in heavily contaminated environments as this can lead to implant failures. With this understanding, we believe that steps need to be taken to safeguard these materials from bacterial colonization. Incorporation of antimicrobial agents, biofilm modifications and bacterial interference agents into devices themselves ought to be further investigated. Newer products and modifications to exiting products may further enhance the benefits of biologic mesh particularly in challenging cases.