Kerry A. Daly

Macrophage phenotype as a predictor of constructive remodeling following the implantation of biologically derived surgical mesh materials

Macrophages have been classified as having plastic phenotypes which exist along a spectrum between M1 (classically activated; pro-inflammatory) and M2 (alternatively activated; regulatory, homeostatic). To date, the effects of polarization towards an M1 or M2 phenotype have been studied largely in the context of response to pathogen or cancer. Recently, M1 and M2 macrophages have been shown to play distinct roles in tissue remodeling following injury. In the present study, the M1/M2 paradigm was utilized to examine the role of macrophages in the remodeling process following implantation of 14 biologically derived surgical mesh materials in the rat abdominal wall. In situ polarization of macrophages responding to the materials was examined and correlated to a quantitative measure of the observed tissue remodeling response to determine whether macrophage polarization is an accurate predictor of the ability of a biologic scaffold to promote constructive tissue remodeling. Additionally the ability of M1 and M2 macrophages to differentially recruit progenitor-like cells in vitro, which are commonly observed to participate in the remodeling of those ECM scaffolds which have a positive clinical outcome, was examined as a possible mechanism underlying the differences in the observed remodeling responses. The results of the present study show that there is a strong correlation between the early macrophage response to implanted materials and the outcome of tissue remodeling. Increased numbers of M2 macrophages and higher ratios of M2:M1 macrophages within the site of remodeling at 14 days were associated with more positive remodeling outcomes (r(2)=0.525-0.686, p<0.05). Further, the results of the present study suggest that the constructive remodeling outcome may be due to the recruitment and survival of different cell populations to the sites of remodeling associated with materials that elicit an M1 vs. M2 response. Both M2 and M0 macrophage conditioned media were shown to have higher chemotactic activities than media conditioned by M1 macrophages (p<0.05). A more thorough understanding of these issues will logically influence the design of next generation biomaterials and the development of regenerative medicine strategies for the formation of functional host tissues.

The Effects of Processing Methods upon Mechanical and Biologic Properties of Porcine Dermal Extracellular Matrix Scaffolds

Biologic materials from various species and tissues are commonly used as surgical meshes or scaffolds for tissue reconstruction. Extracellular matrix (ECM) represents the secreted product of the cells comprising each tissue and organ, and therefore provides a unique biologic material for selected regenerative medicine applications. Minimal disruption of ECM ultrastructure and content during tissue processing is typically desirable. The objective of this study was to systematically evaluate effects of commonly used tissue processing steps upon porcine dermal ECM scaffold composition, mechanical properties, and cytocompatibility. Processing steps evaluated included liming and hot water sanitation, trypsin/SDS/TritonX-100 decellularization, and trypsin/TritonX-100 decellularization. Liming decreased the growth factor and glycosaminoglycan content, the mechanical strength, and the ability of the ECM to support in vitro cell growth (p ≤ 0.05 for all). Hot water sanitation treatment decreased only the growth factor content of the ECM (p ≤ 0.05). Trypsin/SDS/TritonX-100 decellularization decreased the growth factor content and the ability of the ECM to support in vitro cell growth (p ≤ 0.05 for both). Trypsin/Triton X-100 decellularization also decreased the growth factor content of the ECM but increased the ability of the ECM to support in vitro cell growth (p ≤ 0.05 for both). We conclude that processing steps evaluated in the present study affect content, mechanical strength, and/or cytocompatibility of the resultant porcine dermal ECM, and therefore care must be taken in choosing appropriate processing steps to maintain the beneficial effects of ECM in biologic scaffolds.

A hydrogel derived from decellularized dermal extracellular matrix.

The ECM of mammalian tissues has been used as a scaffold to facilitate the repair and reconstruction of numerous tissues. Such scaffolds are prepared in many forms including sheets, powders, and hydrogels. ECM hydrogels provide advantages such as injectability, the ability to fill an irregularly shaped space, and the inherent bioactivity of native matrix. However, material properties of ECM hydrogels and the effect of these properties upon cell behavior are neither well understood nor controlled. The objective of this study was to prepare and determine the structure, mechanics, and the cell response in vitro and in vivo of ECM hydrogels prepared from decellularized porcine dermis and urinary bladder tissues. Dermal ECM hydrogels were characterized by a more dense fiber architecture and greater mechanical integrity than urinary bladder ECM hydrogels, and showed a dose dependent increase in mechanical properties with ECM concentration. In vitro, dermal ECM hydrogels supported greater C2C12 myoblast fusion, and less fibroblast infiltration and less fibroblast mediated hydrogel contraction than urinary bladder ECM hydrogels. Both hydrogels were rapidly infiltrated by host cells, primarily macrophages, when implanted in a rat abdominal wall defect. Both ECM hydrogels degraded by 35 days in vivo, but UBM hydrogels degraded more quickly, and with greater amounts of myogenesis than dermal ECM. These results show that ECM hydrogel properties can be varied and partially controlled by the scaffold tissue source, and that these properties can markedly affect cell behavior.

Biologic scaffold composed of skeletal muscle extracellular matrix.

Biologic scaffolds prepared from the extracellular matrix (ECM) of decellularized mammalian tissues have been shown to facilitate constructive remodeling in injured tissues such as skeletal muscle, the esophagus, and lower urinary tract, among others. The ECM of every tissue has a unique composition and structure that likely has direct effects on the host response and it is plausible that ECM harvested from a given tissue would provide distinct advantages over ECM harvested from nonhomologous tissues. For example, a tissue specific muscle ECM scaffold may be more suitable for constructive remodeling of skeletal muscle than non-homologous ECM tissue sources. The present study describes an enzymatic and chemical decellularization process for isolating skeletal muscle ECM scaffolds using established decellularization criteria and characterized the structure and chemical composition of the resulting ECM. The results were compared to those from a non-muscle ECM derived from small intestine (SIS). Muscle ECM was shown to contain growth factors, glycosaminoglycans, and basement membrane structural proteins which differed from those present in SIS. Myogenic cells survived and proliferated on muscle ECM scaffolds in vitro, and when implanted in a rat abdominal wall injury model in vivo was shown to induce a constructive remodeling response associated with scaffold degradation and myogenesis in the implant area; however, the remodeling outcome did not differ from that induced by SIS by 35 days post surgery. These results suggest that superior tissue remodeling outcomes are not universally dependent upon homologous tissue derived ECM scaffold materials.

Macrophage polarization in response to ECM coated polypropylene mesh

The host response to implanted biomaterials is a highly regulated process that influences device functionality and clinical outcome. Non-degradable biomaterials, such as knitted polypropylene mesh, frequently elicit a chronic foreign body reaction with resultant fibrosis. Previous studies have shown that an extracellular matrix (ECM) hydrogel coating of polypropylene mesh reduces the intensity of the foreign body reaction, though the mode of action is unknown. Macrophage participation plays a key role in the development of the foreign body reaction to biomaterials, and therefore the present study investigated macrophage polarization following mesh implantation. Spatiotemporal analysis of macrophage polarization was conducted in response to uncoated polypropylene mesh and mesh coated with hydrated and dry forms of ECM hydrogels derived from either dermis or urinary bladder. Pro-inflammatory M1 macrophages (CD86+/CD68+), alternatively activated M2 macrophages (CD206+/CD68+), and foreign body giant cells were quantified between 3 and 35 days. Uncoated polypropylene mesh elicited a dominant M1 response at the mesh fiber surface, which was decreased by each ECM coating type beginning at 7 days. The diminished M1 response was accompanied by a reduction in the number of foreign body giant cells at 14 and 35 days, though there was a minimal effect upon the number of M2 macrophages at any time. These results show that ECM coatings attenuate the M1 macrophage response and increase the M2/M1 ratio to polypropylene mesh in vivo.

Polypropylene Surgical Mesh Coated with Extracellular Matrix Mitigates the Host Foreign Body Response

Surgical mesh devices composed of synthetic materials are commonly used for ventral hernia repair. These materials provide robust mechanical strength and are quickly incorporated into host tissue; factors that contribute to reduced hernia recurrence rates. However, such mesh devices cause a foreign body response with the associated complications of fibrosis and patient discomfort. In contrast, surgical mesh devices composed of naturally occurring extracellular matrix (ECM) are associated with constructive tissue remodeling, but lack the mechanical strength of synthetic materials. A method for applying a porcine dermal ECM hydrogel coating to a polypropylene mesh is described herein with the associated effects upon the host tissue response and biaxial mechanical behavior. Uncoated and ECM coated heavy-weight BARD™ Mesh were compared to the light-weight ULTRAPRO™ and BARD™ Soft Mesh devices in a rat partial thickness abdominal defect overlay model. The ECM coated mesh attenuated the pro-inflammatory response compared to all other devices, with a reduced cell accumulation and fewer foreign body giant cells. The ECM coating degraded by 35 days, and was replaced with loose connective tissue compared to the dense collagenous tissue associated with the uncoated polypropylene mesh device. Biaxial mechanical characterization showed that all of the mesh devices were of similar isotropic stiffness. Upon explanation, the light-weight mesh devices were more compliant than the coated or uncoated heavy-weight devices. This study shows that an ECM coating alters the default host response to a polypropylene mesh, but not the mechanical properties in an acute in vivo abdominal repair model.

A rabbit model of peripheral compartment syndrome with associated rhabdomyolysis and a regenerative medicine approach for treatment.

Peripheral compartment syndrome (PCS) has a complex etiology, with limited treatment options and high patient morbidity. Animal models of PCS have been hampered by differences in cross-species anatomy, physiology, and the relative rarity of the naturally occurring syndrome in animals. In the present study, the combination of saline infusion with intermittent crushing of skeletal muscle consistently caused increased intracompartmental pressure, hypocalemia, and hypercreatinine-phophokinasemia, signs diagnostic of PCS. This method was used to evaluate both the standard PCS treatment, specifically a fasciotomy, and a regenerative medicine approach for treatment-consisting of a fasciotomy with local administration of a biologic scaffold material composed of porcine small intestinal submucosa extracellular matrix (SIS-ECM). The use of this SIS-ECM scaffold in conjunction with a fasciotomy was associated with myogenesis and constructive tissue remodeling in the SIS-ECM-treated animals. At 1 and 3 months after treatment innervated muscle tissue was present at the site of injury. No myogenesis was present in the fasciotomy only treated animals. RAM11+ macrophages, which are associated with constructive tissue remodeling, were present within the injury site in the SIS-ECM-treated animals at 1 month. The present study provides a reproducible animal model with which to study PCS, and shows the potential of a regenerative medicine approach to PCS treatment.

In vivo degradation of 14C-labeled porcine dermis biologic scaffold.

Biologic scaffold materials are used for repair and reconstruction of injured or missing tissues. Such materials are often composed of allogeneic or xenogeneic extracellular matrix (ECM) manufactured by decellularization of source tissue, such as dermis. Dermal ECM (D-ECM) has been observed to degrade and remodel in vivo more slowly than other biologic scaffold materials, such as small intestinal submucosa (SIS-ECM). Histologic examination is a common method for evaluating material degradation, but it lacks sensitivity and is subject to observer bias. Utilization of (14)C-proline labeled ECM is a quantitative alternative for measuring degradation of ECM scaffolds. Using both methods, the amount of degradation of D-ECM and SIS-ECM was determined at 2, 4, and 24 weeks post-implantation in a rodent model. Results utilizing (14)C liquid scintillation counting (LSC) analysis showed distinct differences in degradation at the three time points. D-ECM material in situ stayed the same at 76% remaining from 2 to 4 weeks post-implantation, and then decreased to 44% remaining at 24 weeks. In the same time period, implanted SIS-ECM material decreased from 72% to 13% to 0%. Visual examination of device degradation by histology overestimated degradation at 2 weeks and underestimated device degradation at 24 weeks, compared to the (14)C method.

Regenerative Medicine and the Foreign Body Response

The host response, and in particular the innate immune response, is critical to the successful application of tissue engineering to the reconstruction of injured or missing tissues. Cell-based, scaffold-based, and signal molecule-based strategies are utilized in regenerative medicine and each of these approaches elicits a distinct host immune response that has a significant impact upon the downstream outcome. Modulation, but not suppression of the immune component of wound healing appears to be essential for constructive remodeling of tissues and organs. Promotion of a pro-wound healing and anti-inflammatory response, and avoidance of the foreign body reaction is associated with a constructive functional remodeling outcome. While macrophages play a pivotal role in this response, other immune cells and the interactions between all cell types involved in tissue remodeling are also clearly important. The objective of this chapter is to provide an overview of the host response to biomaterials including both the pro-inflammatory and resultant foreign body reaction, and the pro-wound healing, anti-inflammatory response that is associated with constructive remodeling.

A Rodent Model to Evaluate the Tissue Response to a Biological Scaffold When Adjacent to a Synthetic Material.

The use of biologic scaffold materials adjacent to synthetic meshes is commonplace. A prevalent clinical example is two-staged breast reconstruction, where biologic scaffolds are used to provide support and coverage for the inferior aspect of the synthetic expander. However, limited data exist regarding either the kinetics of biologic scaffold integration or the host tissue response to the biologic scaffold materials used for this application or other applications in which such scaffold materials are used. The present study evaluated the temporal host response to a biological scaffold when placed adjacent to a synthetic material. Evaluation criteria included quantification of material contracture and characterization of the host cell response and tissue remodeling events. Results show a decreased thickness of the collagenous tissue layer at biologic scaffold/silicone interface compared to the abdominal wall/silicone interface during the 12-week experimental time course. All test materials were readily incorporated into surrounding host tissue.