Artificial Intelligence Optical Biopsy for Evaluating the Functional State of Wounds.

INTRODUCTION: The clinical characterization of the functional status of active wounds in terms of their driving cellular and molecular biology remains a considerable challenge that currently requires excision via a tissue biopsy. In this pilot study, we use convolutional Siamese neural network (SNN) architecture to predict the functional state of a wound using digital photographs of wounds in a canine model of volumetric muscle loss (VML). METHODS: Digital images of VML injuries and tissue biopsies were obtained in a standardized fashion from an established canine model of VML. Gene expression profiles for each biopsy site were obtained using RNA sequencing. These profiles were converted to functional profiles by a manual review of validated gene ontology databases in which we determined a hierarchical representation of gene functions based on functional specificity. An SNN was trained to regress functional profile expression values, informed by an image segment showing the surface of a small tissue biopsy. RESULTS: The SNN was able to predict the functional expression of a range of functions based with error ranging from ∼5% to ∼30%, with functions that are most closely associated with the early state of wound healing to be those best-predicted. CONCLUSIONS: These initial results suggest promise for further research regarding this novel use of machine learning regression on medical images. The regression of functional profiles, as opposed to specific genes, both addresses the challenge of genetic redundancy and gives a deeper insight into the mechanistic configuration of a region of tissue in wounds.

A Comparative Study of the Resorption and Immune Response for Two Starch-Based Hemostat Powders.

INTRODUCTION: Powder hemostats are valuable adjuncts to minimize intraoperative and postoperative complications. In addition to promotion of rapid coagulation, resorption, and biocompatibility are desirable attributes. Plant starch-based polysaccharide hemostat powders are effective and widely used hemostatic agents, however their source and/or processing can affect characteristics such as in vivo degradability. For example, Arista is a purified/hydrolyzed starch powder that is rapidly resorbed in vivo; whereas PerClot shows slow resorption and preservation of a crystalline form. MATERIALS AND METHODS: In the present study, we compared the cellular response to the hemostatic agents PerClot and Arista both in vitro and in vivo, and used potato starch and urinary bladder extracellular matrix (UBM-ECM) as high crystallinity/slowly resorbable and prohealing controls, respectively. RESULTS: All test articles and their degradation products were cytocompatible in vitro as measured by cell viability and metabolic activity of bone-marrow macrophages. PerClot induced a stronger proinflammatory, M1-like macrophage response in vitro (P < 0.001) than Arista, likely due to differences in source composition. Histologic examination of the in vivo surgical site showed the almost complete degradation of Arista after 12 h (day 0), whereas both PerClot and potato starch were still present at 28 d with crystals identifiable with polarized light microscopy and periodic acid Schiff (PAS) staining. Macrophage phenotype in vivo showed no differences between PerClot and Arista. Collagen deposition and mononuclear cell accumulation consistent with an early foreign body response were present around PerClot and potato starch crystals, whereas no such cell or connective tissue deposition was noted at the site of Arista or UBM-ECM placement.

Optical Biopsy Using a Neural Network to Predict Gene Expression From Photos of Wounds.

BACKGROUND: The clinical characterization of the biological status of complex wounds remains a considerable challenge. Digital photography provides a non-invasive means of obtaining wound information and is currently employed to assess wounds qualitatively. Advances in machine learning (ML) image processing provide a means of identifying "hidden" features in pictures. This pilot study trains a convolutional neural network (CNN) to predict gene expression based on digital photographs of wounds in a canine model of volumetric muscle loss (VML). MATERIALS AND METHODS: Images of volumetric muscle loss injuries and tissue biopsies were obtained in a canine model of VML. A CNN was trained to regress gene expression values as a function of the extracted image segment (color and spatial distribution). Performance of the CNN was assessed in a held-back test set of images using Mean Absolute Percentage Error (MAPE). RESULTS: The CNN was able to predict the gene expression of certain genes based on digital images, with a MAPE ranging from ∼10% to ∼30%, indicating the presence and identification of distinct, and identifiable patterns in gene expression throughout the wound. CONCLUSIONS: These initial results suggest promise for further research regarding this novel use of ML regression on medical images. Specifically, the use of CNNs to determine the mechanistic biological state of a VML wound could aid both the design of future mechanistic interventions and the design of trials to test those therapies. Future work will expand the CNN training and/or test set, with potential expansion to predicting functional gene modules.

Alarmins of the extracellular space.

The ability of the immune system to discriminate between healthy-self, abnormal-self, and non-self has been attributed mainly to alarmins signaling as "danger signals". It is now evident, however, that alarmins are much more complex and can perform specialized functions that can regulate a wide spectrum of processes ranging from propagation of disease to tissue homeostasis. As such, alarmins and their signaling mechanisms are now actively pursued as therapeutic targets. The clinical utility of alarmins requires an understanding of their specific localization. Specifically, many alarmins can function paradoxically depending upon their localization, intra or extracellular. The present review focuses upon alarmin presence and differential expression in the extracellular space versus within the cell and how variation of the localization of alarmins can reveal important mechanistic insights into alarmin functions and their efficacy as biomarkers of disease and therapeutic targets.

Human Bronchial Epithelial Cell Growth on Homologous Versus Heterologous Tissue Extracellular Matrix.

BACKGROUND: Extracellular matrix (ECM) bioscaffolds produced by decellularization of source tissue have been effectively used for numerous clinical applications. However, decellularized tracheal constructs have been unsuccessful due to the immediate requirement of a functional airway epithelium on surgical implantation. ECM can be solubilized to form hydrogels that have been shown to support growth of many different cell types. The purpose of the present study is to compare the ability of airway epithelial cells to attach, form a confluent monolayer, and differentiate on homologous (trachea) and heterologous (urinary bladder) ECM substrates for potential application in full tracheal replacement. MATERIALS AND METHODS: Porcine tracheas and urinary bladders were decellularized. Human bronchial epithelial cells (HBECs) were cultured under differentiation conditions on acellular tracheal ECM and urinary bladder matrix (UBM) bioscaffolds and hydrogels and were assessed by histology and immunolabeling for markers of ciliation, goblet cell formation, and basement membrane deposition. RESULTS: Both trachea and urinary bladder tissues were successfully decellularized. HBEC formed a confluent layer on both trachea and UBM scaffolds and on hydrogels created from these bioscaffolds. Cells grown on tracheal and UBM hydrogels, but not on bioscaffolds, showed positive-acetylated tubulin staining and the presence of mucus-producing goblet cells. Collagen IV immunolabeling showed basement membrane deposition by these cells on the surface of the hydrogels. CONCLUSIONS: ECM hydrogels supported growth and differentiation of HBEC better than decellularized ECM bioscaffolds and show potential utility as substrates for promotion of a mature respiratory epithelium for regenerative medicine applications in the trachea.

Macrophage phenotype in response to ECM bioscaffolds.

Macrophage presence and phenotype are critical determinants of the healing response following injury. Downregulation of the pro-inflammatory macrophage phenotype has been associated with the therapeutic use of bioscaffolds composed of extracellular matrix (ECM), but phenotypic characterization of macrophages has typically been limited to small number of non-specific cell surface markers or expressed proteins. The present study determined the response of both primary murine bone marrow derived macrophages (BMDM) and a transformed human mononuclear cell line (THP-1 cells) to degradation products of two different, commonly used ECM bioscaffolds; urinary bladder matrix (UBM-ECM) and small intestinal submucosa (SIS-ECM). Quantified cell responses included gene expression, protein expression, commonly used cell surface markers, and functional assays. Results showed that the phenotype elicited by ECM exposure (MECM) is distinct from both the classically activated IFNγ+LPS phenotype and the alternatively activated IL-4 phenotype. Furthermore, the BMDM and THP-1 macrophages responded differently to identical stimuli, and UBM-ECM and SIS-ECM bioscaffolds induced similar, yet distinct phenotypic profiles. The results of this study not only characterized an MECM phenotype that has anti-inflammatory traits but also showed the risks and challenges of making conclusions about the role of macrophage mediated events without consideration of the source of macrophages and the limitations of individual cell markers.

Post-Stroke Timing of ECM Hydrogel Implantation Affects Biodegradation and Tissue Restoration.

Extracellular matrix (ECM) hydrogel promotes tissue regeneration in lesion cavities after stroke. However, a bioscaffold's regenerative potential needs to be considered in the context of the evolving pathological environment caused by a stroke. To evaluate this key issue in rats, ECM hydrogel was delivered to the lesion core/cavity at 7-, 14-, 28-, and 90-days post-stroke. Due to a lack of tissue cavitation 7-days post-stroke, implantation of ECM hydrogel did not achieve a sufficient volume and distribution to warrant comparison with the other time points. Biodegradation of ECM hydrogel implanted 14- and 28-days post-stroke were efficiently (80%) degraded by 14-days post-bioscaffold implantation, whereas implantation 90-days post-stroke revealed only a 60% decrease. Macrophage invasion was robust at 14- and 28-days post-stroke but reduced in the 90-days post-stroke condition. The pro-inflammation (M1) and pro-repair (M2) phenotype ratios were equivalent at all time points, suggesting that the pathological environment determines macrophage invasion, whereas ECM hydrogel defines their polarization. Neural cells (neural progenitors, neurons, astrocytes, oligodendrocytes) were found at all time points, but a 90-days post-stroke implantation resulted in reduced densities of mature phenotypes. Brain tissue restoration is therefore dependent on an efficient delivery of a bioscaffold to a tissue cavity, with 28-days post-stroke producing the most efficient biodegradation and tissue regeneration, whereas by 90-days post-stroke, these effects are significantly reduced. Improving our understanding of how the pathological environment influences biodegradation and the tissue restoration process is hence essential to devise engineering strategies that could extend the therapeutic window for bioscaffolds to repair the damaged brain.

The challenge of stress incontinence and pelvic organ prolapse: revisiting biologic mesh materials.

PURPOSE OF REVIEW: The present article reviews the history of mesh-related complications and regulations in SUI and POP repair settings, clinical outcomes associated with the use of biologic and synthetic mesh materials, and novel approaches using modified mesh materials. RECENT FINDINGS: Treatment of pelvic floor disorders, such as stress urinary incontinence (SUI) and pelvic organ prolapse (POP) commonly involves implantation of synthetic surgical mesh materials like polypropylene. Many synthetic mesh materials, however, are associated with a foreign body response upon implantation, which is characterized by fibrotic encapsulation. Complications, including erosion, infections, bleeding, and chronic pain, have led to warnings by regulatory agencies and the recall of several mesh products. To mitigate such complications, biologic mesh materials have been proposed as alternatives for SUI and POP repair. SUMMARY: Clinical outcomes of surgical repair of POP/SUI are similar between biologic and synthetic meshes, but biologic meshes have a lower incidence of adverse effects. Several strategies for modifying or functionalizing biological and synthetic meshes have shown promising results in preclinical studies.

Properties of the Temporomandibular Joint in Growing Pigs.

A subset of temporomandibular joint (TMJ) disorders is attributed to joint degeneration. The pig has been considered the preferred in vivo model for the evaluation of potential therapies for TMJ disorders, and practical considerations such as cost and husbandry issues have favored the use of young, skeletally immature animals. However, the effect of growth on the biochemical and biomechanical properties of the TMJ disk and articulating cartilage has not been examined. The present study investigates the effect of age on the biochemical and biomechanical properties of healthy porcine TMJs at 3, 6, and 9 months of age. DNA, hydroxyproline, and glycosaminoglycan (GAG) content were determined and the disks and condyles were tested in uniaxial unconfined stress relaxation compression from 10% to 30% strain. TMJ disks were further assessed with a tensile test to failure technique, which included the ability to test multiple samples from the same region of an individual disk to minimize the intraspecimen variation. No differences in biochemical properties for the disk or compressive properties at 30% stress relaxation in the disk and condylar cartilage were found. In tension, no differences were observed for peak stress and tensile modulus. The collagen content of the condyle was higher at 9 months than 3 months (p < 0.05), and the GAG content was higher at 9 months than 6 months (p < 0.05). There was a trend of increased compressive instantaneous modulus with age. As such, age-matched controls for growing pigs are probably appropriate for most parameters measured.

Extracellular Matrix for Myocardial Repair.

Multiple strategies have been investigated to restore functional myocardium following injury or disease including the local administration of cytokines or chemokines, stem/progenitor cell therapy, mechanical circulatory support, pharmacologic use, and the use of inductive biomaterials. The use of xenogeneic biologic scaffolds composed of extracellular matrix (ECM) has been shown to facilitate functional restoration of several tissues and organs including the esophagus, skeletal muscle, skin, and myocardium, among others. The present chapter describes the current understanding of specific components of biologic scaffolds composed of ECM, the mechanisms by which ECM bioscaffolds promote constructive cardiac remodeling after injury, determinants of remodeling outcome, and the versatility of ECM as a potential cardiac therapeutic.

Bioscaffold-mediated mucosal remodeling following short-segment colonic mucosal resection.

Precancerous or cancerous lesions of the gastrointestinal tract often require surgical resection via endomucosal resection. Although excision of the colonic mucosa is an effective cancer treatment, removal of large lesions is associated with high morbidity and complications including bleeding, perforation, fistula formation, and/or stricture, contributing to high clinical and economic costs and negatively impacting patient quality of life. The present study investigates the use of a biologic scaffold derived from extracellular matrix (ECM) to promote restoration of the colonic mucosa following short segment mucosal resection. Six healthy dogs were assigned to ECM-treated (tubular ECM scaffold) and mucosectomy only control groups following transanal full circumferential mucosal resection (4 cm in length). The temporal remodeling response was monitored using colonoscopy and biopsy collection. Animals were sacrificed at 6 and 10 wk, and explants were stained with hematoxylin and eosin (H&E), Alcian blue, and proliferating cell nuclear antigen (PCNA) to determine the temporal remodeling response. Both control animals developed stricture and bowel obstruction with no signs of neomucosal coverage after resection. ECM-treated animals showed an early mononuclear cell infiltrate (2 weeks post-surgery) which progressed to columnar epithelium and complex crypt structures nearly indistinguishable from normal colonic architecture by 6 weeks after surgery. ECM scaffold treatment restored colonic mucosa with appropriately located PCNA+ cells and goblet cells. The study shows that ECM scaffolds may represent a viable clinical option to prevent complications associated with endomucosal resection of cancerous lesions in the colon.

Regenerative Medicine Approaches for Age-Related Muscle Loss and Sarcopenia: A Mini-Review.

Sarcopenia is a complex and multifactorial disease that includes a decrease in the number, structure and physiology of muscle fibers, and age-related muscle mass loss, and is associated with loss of strength, increased frailty, and increased risk for fractures and falls. Treatment options are suboptimal and consist of exercise and nutrition as the cornerstone of therapy. Current treatment principles involve identification and modification of risk factors to prevent the disease, but these efforts are of limited value to the elderly individuals currently affected by sarcopenia. The development of new and effective therapies for sarcopenia is challenging. Potential therapies can target one or more of the proposed multiple etiologies such as the loss of regenerative capacity of muscle, age-related changes in the expression of signaling molecules such as growth hormone, IGF-1, myostatin, and other endocrine signaling molecules, and age-related changes in muscle physiology like denervation and mitochondrial dysfunction. The present paper reviews regenerative medicine strategies that seek to restore adequate skeletal muscle structure and function including exogenous delivery of cells and pharmacological therapies to induce myogenesis or reverse the physiologic changes that result in the disease. Approaches that modify the microenvironment to provide an environment conducive to reversal and mitigation of the disease represent a potential regenerative medicine approach that is discussed herein.

Extracellular matrix bioscaffolds in tissue remodeling and morphogenesis.

During normal morphogenesis the extracellular matrix (ECM) influences cell motility, proliferation, apoptosis, and differentiation. Tissue engineers have attempted to harness the cell signaling potential of ECM to promote the functional reconstruction, if not regeneration, of injured or missing adult tissues that otherwise heal by the formation of scar tissue. ECM bioscaffolds, derived from decellularized tissues, have been used to promote the formation of site appropriate, functional tissues in many clinical applications including skeletal muscle, fibrocartilage, lower urinary tract, and esophageal reconstruction, among others. These scaffolds function by the release or exposure of growth factors and cryptic peptides, modulation of the immune response, and recruitment of progenitor cells. Herein, we describe this process of ECM induced constructive remodeling and examine similarities to normal tissue morphogenesis.

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.

Role of the extracellular matrix in whole organ engineering.

End-stage organ failure is a devastating problem with limited therapeutic options. The definitive treatment is orthotropic transplantation, however, there exists a severe shortage of viable donor organs, and this shortage is worsening with an aging demographic and as the number of new cases of organ failure increases. Patients fortunate enough to receive a transplant are required to receive immunosuppressive therapies and can face transplant rejection. The emerging concept of organ engineering may offer a new hope for these patients. Researchers in the field of regenerative medicine and tissue engineering are using three-dimensional whole organ scaffolds composed of allogeneic or xenogeneic extracellular matrix (ECM) for engineering functional tissue suitable for transplantation. Perfusion decellularization is an approach that generates native ECM scaffolds with intact 3D anatomical architecture and vasculature. Decellularized organs provide the ideal transplantable scaffold with all the necessary microstructure and extracellular cues for cell attachment, differentiation, vascularization, and function. The present manuscript will review the role of the ECM in normal development, the concept of ECM tissue specificity, and the effect of processing methods on eventual clinical outcomes. An overview of existing challenges and future directions will also be discussed.

In vitro dose-dependent effects of matrix metalloproteinases on ECM hydrogel biodegradation.

Matrix metalloproteinases (MMPs) cause proteolysis of extracellular matrix (ECM) in tissues affected by stroke. However, little is known about how MMPs degrade ECM hydrogels implanted into stroke cavities to regenerate lost tissue. To establish a structure-function relationship between different doses of individual MMPs and isolate their effects in a controlled setting, an in vitro degradation assay quantified retained urinary bladder matrix (UBM) hydrogel mass as a measure of degradation across time. A rheological characterization indicated that lower ECM concentrations (<4 mg/mL) did not cure completely at 37 °C and had a high fraction of mobile proteins that were easily washed-out. Hydrolysis by dH2O caused a steady 2 % daily decrease in hydrogel mass over 14 days. An acceleration of degradation to 6 % occurred with phosphate buffered saline and artificial cerebrospinal fluid. MMPs induced a dose-dependent increase and within 14 days almost completely (>95 %) degraded the hydrogel. MMP-9 exerted the most significant biodegradation, compared to MMP-3 and -2. To model the in vivo exposure of hydrogel to MMPs, mixtures of MMP-2, -3, and -9, present in the cavity at 14-, 28-, or 90-days post-stroke, revealed that 14- and 28-days mixtures achieved an equivalent biodegradation, but a 90-days mixture exhibited a slower degradation. These results revealed that hydrolysis, in addition to proteolysis, exerts a major influence on the degradation of hydrogels. Understanding the mechanisms of ECM hydrogel biodegradation is essential to determine the therapeutic window for bioscaffold implantation after a stroke, and they are also key to determine optimal degradation kinetics to support tissue regeneration. STATEMENT OF SIGNIFICANCE: After implantation into a stroke cavity, extracellular matrix (ECM) hydrogel promotes tissue regeneration through the degradation of the bioscaffold. However, the process of degradation of an ECM hydrogel remains poorly understood. We here demonstrated in vitro under highly controlled conditions that hydrogel degradation is very dependent on its protein concentration. Lower protein concentration hydrogels were weaker in rheological measurements and particularly susceptible to hydrolysis. The proteolytic degradation of tissue ECM after a stroke is caused by matrix metalloproteinases (MMPs). A dose-dependent MMP-driven biodegradation of ECM hydrogel exceeded the effects of hydrolysis. These results highlight the importance of in vitro testing of putative causes of degradation to gain a better understanding of how these factors affect in vivo biodegradation.

Advances, challenges, and future directions in the clinical translation of ECM biomaterials for regenerative medicine applications.

Extracellular Matrix (ECM) scaffolds and biomaterials have been widely used for decades across a variety of diverse clinical applications and have been implanted in millions of patients worldwide. ECM-based biomaterials have been especially successful in soft tissue repair applications but their utility in other clinical applications such as for regeneration of bone or neural tissue is less well understood. The beneficial healing outcome with the use of ECM biomaterials is the result of their biocompatibility, their biophysical properties and their ability to modify cell behavior after injury. As a consequence of successful clinical outcomes, there has been motivation for the development of next-generation formulations of ECM materials ranging from hydrogels, bioinks, powders, to whole organ or tissue scaffolds. The continued development of novel ECM formulations as well as active research interest in these materials ensures a wealth of possibilities for future clinical translation and innovation in regenerative medicine. The clinical translation of next generation formulations ECM scaffolds faces predictable challenges such as manufacturing, manageable regulatory pathways, surgical implantation, and the cost required to address these challenges. The current status of ECM-based biomaterials, including clinical translation, novel formulations and therapies currently under development, and the challenges that limit clinical translation of ECM biomaterials are reviewed herein.