Neill J. Turner

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.

Functional Skeletal Muscle Formation with a Biologic Scaffold

Biologic scaffolds composed of extracellular matrix (ECM) have been used to reinforce or replace damaged or missing musculotendinous tissues in both preclinical studies and in human clinical applications. However, most studies have focused upon morphologic endpoints and few studies have assessed the in-situ functionality of newly formed tissue; especially new skeletal muscle tissue. The objective of the present study was to determine both the in-situ tetanic contractile response and histomorphologic characteristics of skeletal muscle tissue reconstructed using one of four test articles in a rodent abdominal wall model: 1) porcine small intestinal submucosa (SIS)-ECM; 2) carbodiimide-crosslinked porcine SIS-ECM; 3) autologous tissue; or 4) polypropylene mesh. Six months after surgery, the remodeled SIS-ECM showed almost complete replacement by islands and sheets of skeletal muscle, which generated a similar maximal contractile force to native tissue but with greater resistance to fatigue. The autologous tissue graft was replaced by a mixture of collagenous connective tissue, adipose tissue with fewer islands of skeletal muscle compared to SIS-ECM and a similar fatigue resistance to native muscle. Carbodiimide-crosslinked SIS-ECM and polypropylene mesh were characterized by a chronic inflammatory response and produced little or no measurable tetanic force. The findings of this study show that non-crosslinked xenogeneic SIS scaffolds and autologous tissue are associated with the restoration of functional skeletal muscle with histomorphologic characteristics that resemble native muscle.

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.

Regeneration of skeletal muscle

Skeletal muscle has a robust capacity for regeneration following injury. However, few if any effective therapeutic options for volumetric muscle loss are available. Autologous muscle grafts or muscle transposition represent possible salvage procedures for the restoration of mass and function but these approaches have limited success and are plagued by associated donor site morbidity. Cell-based therapies are in their infancy and, to date, have largely focused on hereditary disorders such as Duchenne muscular dystrophy. An unequivocal need exists for regenerative medicine strategies that can enhance or induce de novo formation of functional skeletal muscle as a treatment for congenital absence or traumatic loss of tissue. In this review, the three stages of skeletal muscle regeneration and the potential pitfalls in the development of regenerative medicine strategies for the restoration of functional skeletal muscle in situ are discussed.

Biologic Scaffold Remodeling in a Dog Model of Complex Musculoskeletal Injury

BACKGROUND Current treatment principles for muscle injuries with volumetric loss have been largely derived from empirical observations. Differences in severity or anatomic location have determinant effects on the tissue remodeling outcome. Biologic scaffolds composed of extracellular matrix (ECM) have been successfully used to restore vascularized, innervated, and contractile skeletal muscle in animal models but limited anatomic locations have been evaluated. The aim of this study was to determine the ability of a xenogeneic ECM scaffold to restore functional skeletal muscle in a canine model of a complex quadriceps injury involving bone, tendon, and muscle. MATERIALS AND METHODS Sixteen dogs were subjected to unilateral resection of the distal third of the vastus lateralis and medial half of the distal third of the vastus medialis muscles including the proximal half of their associated quadriceps tendon. This defect was replaced with a biologic scaffold composed of small intestinal submucosa extracellular matrix (SIS-ECM) and the remodeling response was evaluated at 1, 2, 3, and 6 mo (N = 4 per group). RESULTS The initial remodeling process followed a similar pattern to other studies of ECM-mediated muscle repair with rapid vascularization and migration of myoblasts into the defect site. However, over time the remodeling response resulted in the formation of dense collagenous tissue with islands of muscle in the segments of the scaffold not in contact with bone, and foci of bone and cartilage in the segments that were adjacent to the underlying bone. CONCLUSIONS SIS-ECM was not successful at restoring functional muscle tissue in this model. However, the results also suggest that SIS-ECM may have potential to promote integration of soft and boney tissues when implanted in close apposition to bone.

Matrix-bound nanovesicles within ECM bioscaffolds

Matrix-bound vesicles within ECM bioscaffolds provide mechanistic insight into inductive properties. Biologic scaffold materials composed of extracellular matrix (ECM) have been used in a variety of surgical and tissue engineering/regenerative medicine applications and are associated with favorable constructive remodeling properties including angiogenesis, stem cell recruitment, and modulation of macrophage phenotype toward an anti-inflammatory effector cell type. However, the mechanisms by which these events are mediated are largely unknown. Matrix-bound nanovesicles (MBVs) are identified as an integral and functional component of ECM bioscaffolds. Extracellular vesicles (EVs) are potent vehicles of intercellular communication due to their ability to transfer RNA, proteins, enzymes, and lipids, thereby affecting physiologic and pathologic processes. Formerly identified exclusively in biologic fluids, the presence of EVs within the ECM of connective tissue has not been reported. In both laboratory-produced and commercially available biologic scaffolds, MBVs can be separated from the matrix only after enzymatic digestion of the ECM scaffold material, a temporal sequence similar to the functional activity attributed to implanted bioscaffolds during and following their degradation when used in clinical applications. The present study shows that MBVs contain microRNA capable of exerting phenotypical and functional effects on macrophage activation and neuroblastoma cell differentiation. The identification of MBVs embedded within the ECM of biologic scaffolds provides mechanistic insights not only into the inductive properties of ECM bioscaffolds but also into the regulation of tissue homeostasis.

An acellular biologic scaffold treatment for volumetric muscle loss: results of a 13-patient cohort study

Volumetric muscle loss (VML) is a severe and debilitating clinical problem. Current standard of care includes physical therapy or orthotics, which do not correct underlying strength deficits, and surgical tendon transfers or muscle transfers, which involve donor site morbidity and fall short of restoring function. The results of a 13-patient cohort study are described herein and involve a regenerative medicine approach for VML treatment. Acellular bioscaffolds composed of mammalian extracellular matrix (ECM) were implanted and combined with aggressive and early physical therapy following treatment. Immunolabeling of ultrasound-guided biopsies, and magnetic resonance imaging and computed tomography imaging were performed to analyse the presence of stem/progenitor cells and formation of new skeletal muscle. Force production, range-of-motion and functional task performance were analysed by physical therapists. Electrodiagnostic evaluation was used to analyse presence of innervated skeletal muscle. This study is registered with ClinicalTrials.gov, numbers NCT01292876. In vivo remodelling of ECM bioscaffolds was associated with mobilisation of perivascular stem cells; formation of new, vascularised, innervated islands of skeletal muscle within the implantation site; increased force production; and improved functional task performance when compared with pre-operative performance. Compared with pre-operative performance, by 6 months after ECM implantation, patients showed an average improvement of 37.3% (P<0.05) in strength and 27.1% improvement in range-of-motion tasks (P<0.05). Implantation of acellular bioscaffolds derived from ECM can improve strength and function, and promotes site-appropriate remodelling of VML defects. These findings provide early evidence of bioscaffolding as a viable treatment of VML.

Quantitative multispectral imaging of Herovici's polychrome for the assessment of collagen content and tissue remodelling.

Bioprosthetic devices, constructed from a variety of materials, are routinely implanted in a variety of anatomical locations. Essential to their success is the formation of a non‐destructive interface with the host tissue and appropriate tissue remodelling. Traditionally, the main method of assessing the host–material interface has been qualitative histological evaluation, using pattern recognition and comparative assessment to identify changes in the normal tissue architecture that are characteristic of scar tissue. In the present study, the recently developed technique of multispectral imaging was used to revisit a little‐described histological stain, Herovicis polychrome, which is capable of distinguishing between types I and III collagen. Combined, these techniques allowed quantification of collagen content and distribution of collagen types within a tissue sample. Samples of rat tail and human scar tissue were used to optimize the staining, while comparison with immunolabelled samples was used to develop a reproducible quantification system, based on the specific colour profiles for types I and III collagen. Finally the remodelling of rat abdominal wall defects repaired with crosslinked or non‐crosslinked extracellular matrix scaffolds derived from porcine urinary bladder was assessed with this technique. Compared to standard histological assessment, the combination of multispectral imaging and Herovicis polychrome staining presents a quick, simple, reliable technique that can provide accurate quantification of tissue remodelling and specifically identify the expression and distribution of types I and III collagen. Copyright

Bone marrow–derived cells participate in the long-term remodeling in a mouse model of esophageal reconstruction

BACKGROUND The default response of the esophagus to injury includes inflammation and scar tissue formation often leading to stricture. Biologic scaffolds composed of extracellular matrix (ECM) have been associated with the reconstitution of functional esophageal tissue in preclinical studies and clinical case reports of esophageal mucosal resection, anastomotic reinforcement, and full circumferential replacement. However, the mechanisms responsible for this change in the default response to esophageal injury are not fully understood. METHODS The objective of the present study was to determine whether bone marrow-derived cells (BMCs) participate in the long-term remodeling of ECM scaffolds in the esophageal location in a mouse model. RESULTS BMCs were present in low numbers in remodeling ECM scaffolds. Compared with the untreated control mice, the ECM-implanted animals showed better remodeling of the epithelial layer. CONCLUSIONS BMCs are involved in ECM remodeling process during tissue repair after esophageal injury, but the low numbers argue against any significant involvement in the constructive remodeling process.

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.