Christopher L. Dearth

An Acellular Biologic Scaffold Promotes Skeletal Muscle Formation in Mice and Humans with Volumetric Muscle Loss

Scaffolds composed of cell-free extracellular matrix promote de novo formation of functional skeletal muscle tissue in sites of volumetric muscle loss. Cell-Free Matrix Refills Muscle In traumatic accidents, or even in surgery, large amounts of skeletal muscle can be lost, resulting in pain and loss of function. Although muscle has the ability to regenerate naturally, it cannot refill massive defects, such as those seen in volumetric muscle loss (VML). In response, Sicari and colleagues devised a biomaterial scaffold that can be surgically implanted at the site of VML, encouraging local muscle regeneration and improving function in both mice and humans. The biomaterial used in this study was made up of bladder tissue that had been stripped of cells, leaving behind only the protein scaffold called the extracellular matrix (ECM). Sicari et al. first tested it in a mouse model of VML. In mice treated with ECM, they saw signs of new skeletal muscle formation, characterized by muscle markers desmin and myosin heavy chain, as well as striated (striped) tissue organization. The new muscle also appeared to be innervated, which is necessary for function. The authors translated this preclinical work into a clinical study of five patients with VML and saw outcomes similar to the mice. Six months after ECM implantation at the site of muscle loss, all patients showed signs of new muscle and blood vessels. Three of the five patients showed 20% or greater improvement in limb strength during physical therapy. The two patients without functional changes did report improvements in nonfunctional tasks, such as balance, as well as an improvement in quality of life. Because of the widespread availability and known safety of cell-free ECM-based materials, the approach described by Sicari et al. may translate to regeneration of other human tissues in addition to muscle. Biologic scaffolds composed of naturally occurring extracellular matrix (ECM) can provide a microenvironmental niche that alters the default healing response toward a constructive and functional outcome. The present study showed similarities in the remodeling characteristics of xenogeneic ECM scaffolds when used as a surgical treatment for volumetric muscle loss in both a preclinical rodent model and five male patients. Porcine urinary bladder ECM scaffold implantation was associated with perivascular stem cell mobilization and accumulation within the site of injury, and de novo formation of skeletal muscle cells. The ECM-mediated constructive remodeling was associated with stimulus-responsive skeletal muscle in rodents and functional improvement in three of the five human patients.

The promotion of a constructive macrophage phenotype by solubilized extracellular matrix

The regenerative healing response of injured skeletal muscle is dependent upon a heterogeneous population of responding macrophages, which show a phenotypic transition from the pro-inflammatory M1 to the alternatively activated and constructive M2 phenotype. Biologic scaffolds derived from mammalian extracellular matrix (ECM) have been used for the repair and reconstruction of a variety of tissues, including skeletal muscle, and have been associated with an M2 phenotype and a constructive and functional tissue response. The mechanism(s) behind in-vivo macrophage phenotype transition in skeletal muscle and the enhanced M2:M1 ratio associated with ECM bioscaffold use in-vivo are only partially understood. The present study shows that degradation products from ECM bioscaffolds promote alternatively activated and constructive M2 macrophage polarization in-vitro, which in turn facilitates migration and myogenesis of skeletal muscle progenitor cells.

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.

ECM hydrogel coating mitigates the chronic inflammatory response to polypropylene mesh.

Polypropylene has been used as a surgical mesh material for several decades. This non-degradable synthetic polymer provides mechanical strength, a predictable host response, and its use has resulted in reduced recurrence rates for ventral hernia and pelvic organ prolapse. However, polypropylene and similar synthetic materials are associated with a chronic local tissue inflammatory response and dense fibrous tissue deposition. These outcomes have prompted variations in mesh design to minimize the surface area interface and increase integration with host tissue. In contrast, biologic scaffold materials composed of extracellular matrix (ECM) are rapidly degraded in-vivo and are associated with constructive tissue remodeling and minimal fibrosis. The objective of the present study was to assess the effects of an ECM hydrogel coating on the long-term host tissue response to polypropylene mesh in a rodent model of abdominal muscle injury. At 14 days post implantation, the ECM coated polypropylene mesh devices showed a decreased inflammatory response as characterized by the number and distribution of M1 macrophages (CD86+/CD68+) around mesh fibers when compared to the uncoated mesh devices. At 180 days the ECM coated polypropylene showed decreased density of collagen and amount of mature type I collagen deposited between mesh fibers when compared to the uncoated mesh devices. This study confirms and extends previous findings that an ECM coating mitigates the chronic inflammatory response and associated scar tissue deposition characteristic of polypropylene.

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.

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.

Tissue engineering and regenerative medicine approaches to enhance the functional response to skeletal muscle injury.

The well‐recognized ability of skeletal muscle for functional and structural regeneration following injury is severely compromised in degenerative diseases and in volumetric muscle loss. Tissue engineering and regenerative medicine strategies to support muscle reconstruction have typically been cell‐centric with approaches that involve the exogenous delivery of cells with myogenic potential. These strategies have been limited by poor cell viability and engraftment into host tissue. Alternative approaches have involved the use of biomaterial scaffolds as substrates or delivery vehicles for exogenous myogenic progenitor cells. Acellular biomaterial scaffolds composed of mammalian extracellular matrix (ECM) have also been used as an inductive niche to promote the recruitment and differentiation of endogenous myogenic progenitor cells. An acellular approach, which activates or utilizes endogenous cell sources, obviates the need for exogenous cell administration and provides an advantage for clinical translation. The present review examines the state of tissue engineering and regenerative medicine therapies directed at augmenting the skeletal muscle response to injury and presents the pros and cons of each with respect to clinical translation. Anat Rec, 297:51–64. 2014.

Targeted rehabilitation after extracellular matrix scaffold transplantation for the treatment of volumetric muscle loss.

ABSTRACTRehabilitation therapy is an important aspect of recovery after volumetric muscle loss. However, the traditional rehabilitation approach involves a period of rest and passive loading followed by gradual active loading. Extracellular matrix is a naturally occurring material consisting of structural proteins that provide mechanical strength, structural support, and functional molecules with diverse bioactive properties. There is evidence to suggest that the addition of aggressive regenerative rehabilitation protocols immediately after surgical implantation of an extracellular matrix scaffold to an area of volumetric muscle loss has significant benefits for extracellular matrix remodeling. Rehabilitation exercises likely provide the needed mechanical signals to encourage cell migration and site-specific differentiation in the temporal framework required for constructive remodeling. Herein, the authors review the literature and present an example of an aggressive rehabilitation program implemented immediately after extracellular matrix transplantation into a severely injured quadriceps muscle.

Solubilized extracellular matrix from brain and urinary bladder elicits distinct functional and phenotypic responses in macrophages

Extracellular matrix (ECM) derived from a variety of source tissues has been successfully used to facilitate tissue reconstruction. The recent development of solubilized forms of ECM advances the therapeutic potential of these biomaterials. Isolated, soluble components of ECM and matricryptic peptides have been shown to bias macrophages toward a regulatory and constructive (M2-like) phenotype. However, the majority of studies described thus far have utilized anatomically and morphologically similar gastrointestinal derived ECMs (small intestine, esophagus, urinary bladder, etc.) and a small subset of macrophage markers (CD206, CD86, CCR7) to describe them. The present study evaluated the effect of solubilized ECM derived from molecularly diverse source tissues (brain and urinary bladder) upon primary macrophage phenotype and function. Results showed that solubilized urinary bladder ECM (U-ECM) up-regulated macrophage PGE2 secretion and suppressed traditional pro-inflammatory factor secretion, consistent with an M2-like phenotype. The hyaluronic acid (HA) component in solubilized U-ECM played an important role in mediating this response. Brain ECM (B-ECM) elicited a pro-inflammatory (M1-like) macrophage response and contained almost no HA. These findings suggest that the molecular composition of the source tissue ECM plays an important role in influencing macrophage function and phenotype.

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.