Timothy J. Keane

Consequences of ineffective decellularization of biologic scaffolds on the host response

Biologic scaffold materials composed of extracellular matrix (ECM) are routinely used for a variety of clinical applications. Despite known variations in tissue remodeling outcomes, quantitative criteria by which decellularization can be assessed were only recently described and as a result, the amount of retained cellular material varies widely among commercial products. The objective of this study was to evaluate the consequences of ineffective decellularization on the host response. Three different methods of decellularization were used to decellularize porcine small intestinal ECM (SIS-ECM). The amount of cell remnants was quantified by the amount and fragmentation of DNA within the scaffold materials. The M1/M2 phenotypic polarization profile of macrophages, activated in response to these ECM scaffolds, was assessed in vitro and in vivo using a rodent model of body wall repair. The results show that, in vitro, more aggressive decellularization is associated with a shift in macrophage phenotype predominance from M1 to M2. While this shift was not quantitatively apparent in vivo, notable differences were found in the distribution of M1 vs. M2 macrophages within the various scaffolds. A clear association between macrophage phenotype and remodeling outcome exists and effective decellularization remains an important component in the processing of ECM-based scaffolds.

Methods of tissue decellularization used for preparation of biologic scaffolds and in vivo relevance

Biologic scaffolds composed of extracellular matrix (ECM) are widely used in both preclinical animal studies and in many clinical applications to repair and reconstruct tissues. Recently, 3-dimensional ECM constructs have been investigated for use in whole organ engineering applications. ECM scaffolds are prepared by decellularization of mammalian tissues and the ECM provides natural biologic cues that facilitate the restoration of site appropriate and functional tissue. Preservation of the native ECM constituents (i.e., three-dimensional ultrastructure and biochemical composition) during the decellularization process would theoretically result in the ideal scaffold for tissue remodeling. However, all methods of decellularization invariably disrupt the ECM to some degree. Decellularization of tissues and organs for the production of ECM bioscaffolds requires a balance between maintaining native ECM structure and the removal of cellular materials such as DNA, mitochondria, membrane lipids, and cytosolic proteins. These remnant cellular components can elicit an adverse inflammatory response and inhibit constructive remodeling if not adequately removed. Many variables including cell density, matrix density, thickness, and morphology can affect the extent of tissue and organ decellularization and thus the integrity and physical properties of the resulting ECM scaffold. This review describes currently used decellularization techniques, and the effects of these techniques upon the host response to the material.

Biomaterials for tissue engineering applications

With advancements in biological and engineering sciences, the definition of an ideal biomaterial has evolved over the past 50 years from a substance that is inert to one that has select bioinductive properties and integrates well with adjacent host tissue. Biomaterials are a fundamental component of tissue engineering, which aims to replace diseased, damaged, or missing tissue with reconstructed functional tissue. Most biomaterials are less than satisfactory for pediatric patients because the scaffold must adapt to the growth and development of the surrounding tissues and organs over time. The pediatric community, therefore, provides a distinct challenge for the tissue engineering community.

Preparation and characterization of a biologic scaffold from esophageal mucosa

Biologic scaffolds composed of extracellular matrix (ECM) are commonly used to facilitate a constructive remodeling response in several types of tissue, including the esophagus. Surgical manipulation of the esophagus is often complicated by stricture, but preclinical and clinical studies have shown that the use of an ECM scaffold can mitigate stricture and promote a constructive outcome after resection of full circumference esophageal mucosa. Recognizing the potential benefits of ECM derived from homologous tissue (i.e., site-specific ECM), the objective of the present study was to prepare, characterize, and assess the in-vivo remodeling properties of ECM from porcine esophageal mucosa. The developed protocol for esophageal ECM preparation is compliant with previously established criteria of decellularization and results in a scaffold that maintains important biologic components and an ultrastructure consistent with a basement membrane complex. Perivascular stem cells remained viable when seeded upon the esophageal ECM scaffold in-vitro, and the in-vivo host response showed a pattern of constructive remodeling when implanted in soft tissue.

The host response to allogeneic and xenogeneic biological scaffold materials

The clinical use of biological scaffold materials has become commonplace. Such scaffolds are composed of extracellular matrix (ECM), or components of ECM, derived from allogeneic or xenogeneic tissues. Such scaffold materials vary widely in their source tissue, processing methods and sterilization methods. The success or failure of an ECM scaffold for a given application is dependent on the host response following implantation; a response that is largely mediated by the innate immune system and which is influenced by a numerous factors, including the processing methods used in the preparation of biological scaffolds. The present paper reviews various aspects of the host response to biological scaffolds and factors that affect this response. In addition, some of the logistical, regulatory and reconstructive implications associated with the use of biological scaffolds are discussed. Copyright

Concentration-dependent rheological properties of ECM hydrogel for intracerebral delivery to a stroke cavity

UNLABELLED Biomaterials composed of mammalian extracellular matrix (ECM) promote constructive tissue remodeling with minimal scar tissue formation in many anatomical sites. However, the optimal shape and form of ECM scaffold for each clinical application can vary markedly. ECM hydrogels have been shown to promote chemotaxis and differentiation of neuronal stem cells, but minimally invasive delivery of such scaffold materials to the central nervous system (CNS) would require an injectable form. These ECM materials can be manufactured to exist in fluid phase at room temperature, while forming hydrogels at body temperature in a concentration-dependent fashion. Implantation into the lesion cavity after a stroke could hence provide a means to support endogenous repair mechanisms. Herein, we characterize the rheological properties of an ECM hydrogel composed of urinary bladder matrix (UBM) that influence its delivery and in vivo interaction with host tissue. There was a notable concentration-dependence in viscosity, stiffness, and elasticity; all characteristics important for minimally invasive intracerebral delivery. An efficient MRI-guided injection with drainage of fluid from the cavity is described to assess in situ hydrogel formation and ECM retention at different concentrations (0, 1, 2, 3, 4, and 8mg/mL). Only ECM concentrations >3mg/mL gelled within the stroke cavity. Lower concentrations were not retained within the cavity, but extensive permeation of the liquid phase ECM into the peri-infarct area was evident. The concentration of ECM hydrogel is hence an important factor affecting gelation, host-biomaterial interface, as well intra-lesion distribution. STATEMENT OF SIGNIFICANCE Extracellular matrix (ECM) hydrogel promotes constructive tissue remodeling in many tissues. Minimally invasive delivery of such scaffold materials to the central nervous system (CNS) would require an injectable form that exists in fluid phase at room temperature, while forming hydrogels at body temperature in a concentration-dependent fashion. We here report the rheological characterization of an injectable ECM hydrogel and its concentration-dependent delivery into a lesion cavity formed after a stroke based on MRI-guidance. The concentration of ECM determined its retention within the cavity or permeation into tissue and hence influenced its interaction with the host brain. This study demonstrates the importance of understanding the structure-function relationship of biomaterials to guide particular clinical applications.

Intestinal stem cell growth and differentiation on a tubular scaffold with evaluation in small and large animals

AIMS To investigate the growth and differentiation of intestinal stem cells on a novel tubular scaffold in vitro and in vivo. MATERIALS & METHODS Intestinal progenitor cells from mice or humans were cultured with myofibroblasts, macrophages and/or bacteria, and evaluated in mice via omental implantation. Mucosal regeneration was evaluated in dogs after rectal mucosectomy followed by scaffold implantation. RESULTS Intestinal progenitor cells differentiated into crypt-villi structures on the scaffold. Differentiation and scaffold coverage was enhanced by coculture with myofibroblasts, macrophages and probiotic bacteria, while the implanted scaffolds enhanced mucosal regeneration in the dog rectum. CONCLUSION Intestinal stem cell growth and differentiation on a novel tubular scaffold is enhanced through addition of cellular and microbial components, as validated in mice and dogs.

The effect of terminal sterilization on the material properties and in vivo remodeling of a porcine dermal biologic scaffold.

UNLABELLED Biologic scaffolds composed of extracellular matrix are commonly used in a variety of surgical procedures. The Food and Drug Administration typically regulates biologic scaffolds as medical devices, thus requiring terminal sterilization prior to clinical use. However, to date, no consensus exists for the most effective yet minimally destructive sterilization protocol for biologic scaffold materials. The objective of the present study was to characterize the effect of ethylene oxide, gamma irradiation and electron beam (e-beam) irradiation on the material properties and the elicited in vivo remodeling response of a porcine dermal biologic scaffold. Outcome measures included biochemical, structural, and mechanical properties as well as cytocompatibility in vitro. In vivo evaluation utilized a rodent model to examine the host response to the materials following 7, 14, and 35 days. The host response to each experimental group was determined by quantitative histologic methods and by immunolabeling for macrophage polarization (M1/M2). In vitro results show that increasing irradiation dosage resulted in a dose dependent decrease in mechanical properties compared to untreated controls. Ethylene oxide-treated porcine dermal ECM resulted in decreased DNA content, extractable total protein, and bFGF content compared to untreated controls. All ETO treated, gamma irradiated, and e-beam irradiated samples had similar cytocompatibility scores in vitro. However, in vivo results showed that increasing dosages of e-beam and gamma irradiation elicited an increased rate of degradation of the biologic scaffold material following 35 days. STATEMENT OF SIGNIFICANCE The FDA typically regulates biologic scaffolds derived from mammalian tissues as medical devices, thus requiring terminal sterilization prior to clinical use. However, there is little data and no consensus for the most effective yet minimally destructive sterilization protocol for such materials. The present study characterized the effect of common sterilization methods: ethylene oxide, gamma irradiation and electron beam irradiation on the material properties and the elicited in vivo remodeling response of a porcine dermal biologic scaffold. The results of the study will aid in the meaningful selection of sterilization methods for biologic scaffold materials.

Inhibition of COX1/2 alters the host response and reduces ECM scaffold mediated constructive tissue remodeling in a rodent model of skeletal muscle injury.

UNLABELLED Extracellular matrix (ECM) has been used as a biologic scaffold material to both reinforce the surgical repair of soft tissue and serve as an inductive template to promote a constructive tissue remodeling response. Success of such an approach is dependent on macrophage-mediated degradation and remodeling of the biologic scaffold. Macrophage phenotype during these processes is a predictive factor of the eventual remodeling outcome. ECM scaffolds have been shown to promote an anti-inflammatory or M2-like macrophage phenotype in vitro that includes secretion of downstream products of cycolooxygenases 1 and 2 (COX1/2). The present study investigated the effect of a common COX1/2 inhibitor (Aspirin) on macrophage phenotype and tissue remodeling in a rodent model of ECM scaffold treated skeletal muscle injury. Inhibition of COX1/2 reduced the constructive remodeling response by hindering myogenesis and collagen deposition in the defect area. The inhibited response was correlated with a reduction in M2-like macrophages in the defect area. The effects of Aspirin on macrophage phenotype were corroborated using an established in vitro macrophage model which showed a reduction in both ECM induced prostaglandin secretion and expression of a marker of M2-like macrophages (CD206). These results raise questions regarding the common peri-surgical administration of COX1/2 inhibitors when biologic scaffold materials are used to facilitate muscle repair/regeneration. STATEMENT OF SIGNIFICANCE COX1/2 inhibitors such as nonsteroidal anti-inflammatory drugs (NSAIDs) are routinely administered post-surgically for analgesic purposes. While COX1/2 inhibitors are important in pain management, they have also been shown to delay or diminish the healing process, which calls to question their clinical use for treating musculotendinous injuries. The present study aimed to investigate the influence of a common NSAID, Aspirin, on the constructive remodeling response mediated by an ECM scaffold (UBM) in a rat skeletal muscle injury model. The COX1/2 inhibitor, Aspirin, was found to mitigate the ECM scaffold-mediated constructive remodeling response both in an in vitro co-culture system and an in vivo rat model of skeletal muscle injury. The results presented herein provide data showing that NSAIDs may significantly alter tissue remodeling outcomes when a biomaterial is used in a regenerative medicine/tissue engineering application. Thus, the decision to prescribe NSAIDs to manage the symptoms of inflammation post-ECM scaffold implantation should be carefully considered.

Restoring Mucosal Barrier Function and Modifying Macrophage Phenotype with an Extracellular Matrix Hydrogel: Potential Therapy for Ulcerative Colitis

Background and Aims Despite advances in therapeutic options, more than half of all patients with ulcerative colitis [UC] do not achieve long-term remission, many require colectomy, and the disease still has a marked negative impact on quality of life. Extracellular matrix [ECM] bioscaffolds facilitate the functional repair of many soft tissues by mechanisms that include mitigation of pro-inflammatory macrophage phenotype and mobilization of endogenous stem/progenitor cells. The aim of the present study was to determine if an ECM hydrogel therapy could influence outcomes in an inducible rodent model of UC. Methods The dextran sodium sulphate [DSS]-colitis model was used in male Sprague Dawley rats. Animals were treated via enema with an ECM hydrogel and the severity of colitis was determined by clinical and histological criteria. Lamina propria cells were isolated and the production of inflammatory mediators was quantified. Mucosal permeability was assessed in vivo by administering TRITC-dextran and in vitro using transepithelial electrical resistance [TEER]. Results ECM hydrogel therapy accelerated healing and improved outcome. The hydrogel was adhesive to colonic tissue, which allowed for targeted delivery of the therapy, and resulted in a reduction in clinical and histological signs of disease. ECM hydrogel facilitated functional improvement of colonic epithelial barrier function and the resolution of the pro-inflammatory state of tissue macrophages. Conclusions The present study shows that a non-surgical and non-pharmacological ECM-based therapy can abate DSS-colitis not by immunosuppression but by promoting phenotypic change in local macrophage phenotype and rapid replacement of the colonic mucosal barrier.