Multi-Scale Analysis of the Composition, Structure, and Function of Decellularized Extracellular Matrix for Human Skin and Wound Healing Models.

The extracellular matrix (ECM) is a complex mixture of structural proteins, proteoglycans, and signaling molecules that are essential for tissue integrity and homeostasis. While a number of recent studies have explored the use of decellularized ECM (dECM) as a biomaterial for tissue engineering, the complete composition, structure, and mechanics of these materials remain incompletely understood. In this study, we performed an in-depth characterization of skin-derived dECM biomaterials for human skin equivalent (HSE) models. The dECM materials were purified from porcine skin, and through mass spectrometry profiling, we quantified the presence of major ECM molecules, including types I, III, and VI collagen, fibrillin, and lumican. Rheological analysis demonstrated the sol-gel and shear-thinning properties of dECM materials, indicating their physical suitability as a tissue scaffold, while electron microscopy revealed a complex, hierarchical structure of nanofibers in dECM hydrogels. The dECM materials were compatible with advanced biofabrication techniques, including 3D printing within a gelatin microparticle support bath, printing with a sacrificial material, or blending with other ECM molecules to achieve more complex compositions and structures. As a proof of concept, we also demonstrate how dECM materials can be fabricated into a 3D skin wound healing model using 3D printing. Skin-derived dECM therefore represents a complex and versatile biomaterial with advantageous properties for the fabrication of next-generation HSEs.

Fabrication of Human Skin Equivalents Using Decellularized Extracellular Matrix.

There is a growing demand for in vitro models of human tissues that recapitulate the complex structures and functions found in vivo, and the biomaterials that support these physiologically relevant models are essential underpinning technologies. Here, we present an optimized protocol for generating human skin equivalents (HSEs) using a dermal matrix isolated from decellularized porcine skin. The decellularized extracellular matrix (dECM) contains a complex mixture of fibrillar collagens and matrisomal proteins that mimic native skin and can be produced in large quantities. The procedure for decellularization, digestion, and solubilization of the dECM is described in detail. In addition, we provide instructions for how to construct a three-dimensional HSE model using the dECM as the dermal support matrix for human keratinocytes and dermal fibroblasts. Recent studies from our laboratory have shown that HSEs generated using porcine dECM display improved epidermal differentiation and stratification compared to existing protocols using type I collagen gels. Thus, dECM-based biomaterials are a useful tool for replicating human skin physiology in vitro and developing advanced human skin models for therapeutic discovery and testing. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of decellularized extracellular matrix from porcine skin Basic Protocol 2: Generation of human skin equivalents.

Biological Effects of Phosphocitrate on Osteoarthritic Articular Chondrocytes.

BACKGROUND: Phosphocitrate (PC) inhibits osteoarthritis (OA) in Hartley guinea pigs. However, the underlying molecular mechanisms remain poorly understood. OBJECTIVE: This study sought to examine the biological effect of PC on OA chondrocytes and test the hypothesis that PC may exert its OA disease modifying effect, in part, by inhibiting the expression of genes implicated in OA disease process and stimulating the production of extracellular matrices. METHOD: OA chondrocytes were cultured in the absence or presence of PC. Total RNA was extracted and subjected to microarray analyses. The effect of PC on proliferation and chondrocyte-mediated calcification were examined in monolayer culture. The effect of PC on the production of extracellular matrices was examined in micromass culture. RESULTS: PC downregulated the expression of numerous genes classified in proliferation and apoptosis while upregulating the expression of many genes classified in transforming growth factor-ß (TGF-ß) receptor signaling pathway and ossification. PC also downregulated the expressions of many genes classified in inflammatory response and Wnt receptor signaling pathways. Consistent with its effect on the expression of genes classified in proliferation, ossification, and skeletal development, PC inhibited the proliferation of OA chondrocytes and chondrocyte-mediated calcification while stimulating the production of extracellular matrices. CONCLUSION: PC may exert its OA disease modifying effect, in part, through a crystal-independent mechanism or by inhibiting the expressions of many genes implicated in OA disease process, and at the same time, stimulating the expression of genes implicated in chondroprotection and production of extracellular matrices.

Fibroblast-like synoviocytes induce calcium mineral formation and deposition.

Calcium crystals are present in the synovial fluid of 65%-100% patients with osteoarthritis (OA) and 20%-39% patients with rheumatoid arthritis (RA). This study sought to investigate the role of fibroblast-like synoviocytes (FLSs) in calcium mineral formation. We found that numerous genes classified in the biomineral formation process, including bone gamma-carboxyglutamate (gla) protein/osteocalcin, runt-related transcription factor 2, ankylosis progressive homolog, and parathyroid hormone-like hormone, were differentially expressed in the OA and RA FLSs. Calcium deposits were detected in FLSs cultured in regular medium in the presence of ATP and FLSs cultured in chondrogenesis medium in the absence of ATP. More calcium minerals were deposited in the cultures of OA FLSs than in the cultures of RA FLSs. Examination of the micromass stained with nonaqueous alcoholic eosin indicated the presence of birefringent crystals. Phosphocitrate inhibited the OA FLSs-mediated calcium mineral deposition. These findings together suggest that OA FLSs are not passive bystanders but are active players in the pathological calcification process occurring in OA and that potential calcification stimuli for OA FLSs-mediated calcium deposition include ATP and certain unidentified differentiation-inducing factor(s). The OA FLSs-mediated pathological calcification process is a valid target for the development of disease-modifying drug for OA therapy.

Aquaporin-4 antibody negative recurrent isolated optic neuritis: clinical evidence for disease heterogeneity.

Recurrent optic neuritis is frequently observed in multiple sclerosis (MS) and is a typical finding in neuromyelitis optica (NMO). Patients that lack further evidence of demyelinating disease are diagnosed with RION (recurrent isolated optic neuritis) or CRION (chronic relapsing inflammatory neuropathy) if they require immunosuppressive therapy to prevent further relapses. The etiology and disease course of this rare condition are not well defined. We studied a series of 10 patients who presented with recurrent episodes of isolated optic neuritis (ON, n=57) and were followed over a median of 3.5 years. Visual acuity was severely reduced at the nadir of the disease (20/200 to 20/800). All patients had MRI non-diagnostic for MS/NMO and were aquaporin-4 antibody negative. Six patients fulfilled the CRION criteria. In two of these a single ON followed by a long disease-free interval preceded development of CRION for years, suggesting the conversion of an initially "benign" isolated ON into the chronic relapsing course. Cerebrospinal fluid (CSF) analysis revealed mild pleocytosis in 5 patients, identical oligoclonal bands in serum and CSF were observed in 2 patients, while the others remained negative. In conclusion, recurrent ON is a disease entity that requires aggressive glucocorticoid and eventually long-term immunosuppressive therapy to prevent substantial visual impairment.

Phosphocitrate is potentially a disease-modifying drug for noncrystal-associated osteoarthritis.

Phosphocitrate (PC), a calcification inhibitor, inhibits the development of crystal-associated osteoarthritis (OA) in Hartley guinea pigs. However, the molecular mechanisms underlying its disease-modifying effect remain elusive. This study sought to test the hypothesis that PC has calcium crystal-independent biological activities which are, at least in part, responsible for its disease-modifying activity. We found that PC inhibited the proliferation of OA fibroblast-like synoviocytes in the absence of calcium crystals. Consistent with its effect on cell proliferation, PC downregulated the expression of numerous genes classified in cell proliferation. PC also downregulated the expression of many genes classified in angiogenesis and inflammatory response including prostaglandin-endoperoxide synthase 2, interleukin-1 receptor, type I, and chemokine (C-C motif) ligand 2. In contrast, PC upregulated the expression of many genes classified in musculoskeletal tissue development, including aggrecan, type I collagen, and insulin-like growth factor binding protein 5. These findings suggest that PC is not only a promising disease-modifying drug for crystal-associated OA but also for noncrystal-associated OA.