Ovine Forestomach Matrix in the Surgical Management of Complex Lower-Extremity Soft-Tissue Defects.

BACKGROUND: Chronic lower-extremity defects may lead to major amputations and have severe consequences on patient quality of life and mortality. Dermal matrices have become part of the reconstructive ladder and are often deployed in these scenarios to quickly build neodermis, especially in volumetric defects over exposed bone and tendon initially, to allow for subsequent closure by means of split-thickness skin grafting (STSG) or secondary intention. Ovine forestomach matrix (OFM) is a decellularized extracellular matrix (ECM) bioscaffold available in both sheet and particulate forms that can be used as a dermal matrix in various soft-tissue reconstruction procedures. METHODS: This retrospective case series evaluated the use of OFM products in the surgical reconstruction of 50 cases (n = 50) comprised of challenging lower-extremity defects from seven healthcare centers. Patient records were reviewed to identify comorbidities, defect cause, defect size, presence of exposed structures, Centers for Disease Control and Prevention contamination score, Wagner grade, OFM graft use, time to 100% granulation tissue, STSG use, overall time to heal, and postoperative complications. The primary study outcomes were time (days) to 100% granulation tissue formation, with secondary outcomes including overall time to wound closure (weeks), STSG take at 1 week, and complications. RESULTS: The results of this case series demonstrate OFM as a clinically effective treatment in the surgical management of complex lower-extremity soft-tissue defects with exposed structures in patients with multiple comorbidities. One application of OFM products was effective in regenerating well-vascularized neodermis, often in the presence of exposed structures, with a mean time to 100% granulation of 26.0 ± 22.2 days. CONCLUSIONS: These data support the use of OFM as a safe, cost-effective, and clinically effective treatment option for coverage in complex soft-tissue wounds, including exposed vital structures, and to shorten the time to definitive wound closure in complicated patient populations.

Influence of advanced wound matrices on observed vacuum pressure during simulated negative pressure wound therapy.

Biomaterials and negative pressure wound therapy (NPWT) are treatment modalities regularly used together to accelerate soft-tissue regeneration. This study evaluated the impact of the design and composition of commercially available collagen-based matrices on the observed vacuum pressure delivered under NPWT using a custom test apparatus. Specifically, testing compared the effect of the commercial products; ovine forestomach matrix (OFM), collagen/oxidized regenerated cellulose (collagen/ORC) and a collagen-based dressing (CWD) on the observed vacuum pressure. OFM resulted in an ∼50% reduction in the observed target vacuum pressure at 75 mmHg and 125 mmHg, however, this effect was mitigated to a ∼0% reduction when fenestrations were introduced into the matrix. Both collagen/ORC and CWD reduced the observed vacuum pressure at 125 mmHg (∼15% and ∼50%, respectively), and this was more dramatic when a lower vacuum pressure of 75 mmHg was delivered (∼20% and ∼75%, respectively). The reduced performance of the reconstituted collagen products is thought to result from the gelling properties of these products that may cause occlusion of the delivered vacuum to the wound bed. These findings highlight the importance of in vitro testing to establish the impact of adjunctive therapies on NPWT, where effective delivery of vacuum pressure is paramount to the efficacy of this therapy.

Retrospective real-world comparative effectiveness of ovine forestomach matrix and collagen/ORC in the treatment of diabetic foot ulcers.

The retrospective pragmatic real-world data (RWD) study compared the healing outcomes of diabetic foot ulcers (DFUs) treated with either ovine forestomach matrix (OFM) (n = 1150) or collagen/oxidised regenerated cellulose (ORC) (n = 1072) in out-patient wound care centres. Median time to wound closure was significantly (P = .0015) faster in the OFM group (14.6 ± 0.5 weeks) relative to the collagen/ORC group (16.4 ± 0.7). A sub-group analysis was performed to understand the relative efficacy in DFUs requiring longer periods of treatment and showed that DFUs treated with OFM healed up to 5.3 weeks faster in these challenging wounds. The percentage of wounds closed at 36 weeks was significantly improved in OFM treated DFUs relative to the collagen/ORC. A Cox proportional hazards analysis showed OFM-treated wounds had a 18% greater probability of healing versus wounds managed with collagen/ORC, and the probability increased to 21% when the analysis was adjusted for multiple variables. This study represents the first large retrospective RWD analysis comparing OFM and collagen/ORC and supports the clinical efficacy of OFM in the treatment of DFUs.

Further structural characterization of ovine forestomach matrix and multi-layered extracellular matrix composites for soft tissue repair.

Decellularized extracellular matrix (dECM)-based biomaterials are of great clinical utility in soft tissue repair applications due to their regenerative properties. Multi-layered dECM devices have been developed for clinical indications where additional thickness and biomechanical performance are required. However, traditional approaches to the fabrication of multi-layered dECM devices introduce additional laminating materials or chemical modifications of the dECM that may impair the biological functionality of the material. Using an established dECM biomaterial, ovine forestomach matrix, a novel method for the fabrication of multi-layered dECM constructs has been developed, where layers are bonded via a physical interlocking process without the need for additional bonding materials or detrimental chemical modification of the dECM. The versatility of the interlocking process has been demonstrated by incorporating a layer of hyaluronic acid to create a composite material with additional biological functionality. Interlocked composite devices including hyaluronic acid showed improved in vitro bioactivity and moisture retention properties.

Correction: A novel chemotactic factor derived from the extracellular matrix protein decorin recruits mesenchymal stromal cells in vitro and in vivo.

[This corrects the article DOI: 10.1371/journal.pone.0235784.].

A novel chemotactic factor derived from the extracellular matrix protein decorin recruits mesenchymal stromal cells in vitro and in vivo.

Soft tissue is composed of cells surrounded by an extracellular matrix that is made up of a diverse array of intricately organized proteins. These distinct components work in concert to maintain homeostasis and respond to tissue damage. During tissue repair, extracellular matrix proteins and their degradation products are known to influence physiological processes such as angiogenesis and inflammation. In this study we developed a discovery platform using a decellularized extracellular matrix biomaterial to identify new chemotrophic factors derived from the extracellular matrix. An in vitro culture of RAW.264 macrophage cells with the biomaterial ovine forestomach matrix led to the identification of a novel ~12 kDa chemotactic factor, termed 'MayDay', derived from the N-terminal 31-188 sequence of decorin. The recombinant MayDay protein was shown to be a chemotactic agent for mesenchymal stromal cells in vitro and in vivo. We hypothesize that the macrophage-induced cleavage of decorin, via MMP-12, leads to the release of the chemotactic molecule MayDay, that in turn recruits cells to the site of damaged tissue.

Ionic silver functionalized ovine forestomach matrix - a non-cytotoxic antimicrobial biomaterial for tissue regeneration applications.

BACKGROUND: Antimicrobial technologies, including silver-containing medical devices, are increasingly utilized in clinical regimens to mitigate risks of microbial colonization. Silver-functionalized resorbable biomaterials for use in wound management and tissue regeneration applications have a narrow therapeutic index where antimicrobial effectiveness may be outweighed by adverse cytotoxicity. We examined the effects of ionic silver functionalization of an extracellular matrix (ECM) biomaterial derived from ovine forestomach (OFM-Ag) in terms of material properties, antimicrobial effectiveness and cytotoxicity profile. METHODS: Material properties of OFM-Ag were assessed by via biochemical analysis, microscopy, atomic absorption spectroscopy (AAS) and differential scanning calorimetry. The silver release profile of OFM-Ag was profiled by AAS and antimicrobial effectiveness testing utilized to determine the minimum effective concentration of silver in OFM-Ag in addition to the antimicrobial spectrum and wear time. Biofilm prevention properties of OFM-Ag in comparison to silver containing collagen dressing materials was quantified via in vitro crystal violet assay using a polymicrobial model. Toxicity of ionic silver, OFM-Ag and silver containing collagen dressing materials was assessed toward mammalian fibroblasts using elution cytoxicity testing. RESULTS: OFM-Ag retained the native ECM compositional and structural characteristic of non-silver functionalized ECM material while imparting broad spectrum antimicrobial effectiveness toward 11 clinically relevant microbial species including fungi and drug resistant strains, maintaining effectiveness over a wear time duration of 7-days. OFM-Ag demonstrated significant prevention of polymicrobial biofilm formation compared to non-antimicrobial and silver-containing collagen dressing materials. Where silver-containing collagen dressing materials exhibited cytotoxic effects toward mammalian fibroblasts, OFM-Ag was determined to be non-cytotoxic, silver elution studies indicated sustained retention of silver in OFM-Ag as a possible mechanism for the attenuated cytotoxicity. CONCLUSIONS: This work demonstrates ECM biomaterials may be functionalized with silver to favourably shift the balance between detrimental cytotoxic potential and beneficial antimicrobial effects, while preserving the ECM structure and function of utility in tissue regeneration applications.

Functional Insights from the Proteomic Inventory of Ovine Forestomach Matrix.

Ovine forestomach matrix (OFM) is a decellularized extracellular matrix (dECM) biomaterial that serves as a scaffold for remodeling damaged soft tissue. dECM biomaterials are used in a variety of clinical applications, and their regenerative capacity is encoded not only in their biophysical properties but also in their molecular diversity. In this study, the proteome of OFM was characterized via both targeted and global mass spectrometry (MS) with the use of heavy isotope labeled (SIL) internal standards. Proteins were identified following either chemical digestion or extraction using saline or guanidine hydrochloride, followed by high resolution size exclusion chromatography. Identified proteins were annotated using the matrisome database and molecular function using the gene ontology database. The characterization identified 153 unique matrisome proteins, including 25 collagens, 58 glycoproteins, 12 proteoglycans, 13 ECM affiliated proteins, 20 ECM regulators, and 23 secreted factors. This inventory represents a comprehensive array of matrix proteins that are retained in OFM after processing. The diversity of proteins identified may contribute to OFM's remodeling capacity in clinical applications.

Collagen Fibril Response to Strain in Scaffolds from Ovine Forestomach for Tissue Engineering.

Scaffold biomaterials are typically applied surgically as reinforcement for weakened or damaged tissue, acting as substrates on which healing tissue can grow. Natural extracellular matrix (ECM) materials consisting mainly of collagen are often used for this purpose, but are anisotropic. Ovine forestomach matrix (OFM) ECM was exposed to increasing strain and synchrotron-based SAXS diffraction patterns and revealed that the collagen fibrils within underwent changes in orientation, orientation index (a measure of isotropy), and extension. Response to the strain depended on the direction the collagen fibrils were oriented. When the ECM was stretched in the direction of collagen fibril orientation, the fibrils become more oriented and begin to take up the strain immediately (as shown by the increased d-spacing). Stretch applied perpendicular to dominant fibril direction caused the fibrils to initially become less oriented as they were pulled away from the original direction, and less force was initially transmitted along the length of the fibrils (i.e., the d-spacing changed less). SAXS analysis of OFM and the starting raw tissue showed there is no difference in the structural arrangement of the collagen fibrils. Understanding the directional structural response of these materials under strain may influence how surgeons select and place the materials in use.

Use of versican variant V3 and versican antisense expression to engineer cultured human skin containing increased content of insoluble elastin.

Skin substitutes for repair of dermal wounds are deficient in functional elastic fibres. We report that the content of insoluble elastin in the dermis of cultured human skin can be increased though the use of two approaches that enhance elastogenesis by dermal fibroblasts, forced expression of versican variant V3, which lacks glycosaminoglycan (GAG) chains, and forced expression of versican antisense to decrease levels of versican variant V1 with GAG chains. Human dermal fibroblasts transduced with V3 or anti-versican were cultured under standard conditions over a period of 4 weeks to produce dermal sheets, with growth enhanced though multiple seedings for the first 3 weeks. Human keratinocytes, cultured in supplemented media, were added to the 4-week dermal sheets and the skin layer cultured for a further week. At 5 weeks, keratinocytes were multilayered and differentiated, with desmosome junctions thoughout and keratin deposits in the upper squamous layers. The dermal layer was composed of layered fibroblasts surrounded by extracellular matrix of collagen bundles and, in control cultures, small scattered elastin deposits. Forced expression of V3 and versican antisense slowed growth, decreased versican V1 expression, increased tropoelastin expression and/or the deposition of large aggregates of insoluble elastin in the dermal layer, and increased tissue stiffness, as measured by nano-indentation. Skin sheets were also cultured on Endoform Dermal Template™, the biodegradable wound dressing made from the lamina propria of sheep foregut. Skin structure and the enhanced deposition of elastin by forced expression of V3 and anti-versican were preserved on this supportive substrate. Copyright © 2014 John Wiley & Sons, Ltd.

Ovine forestomach matrix biomaterial is a broad spectrum inhibitor of matrix metalloproteinases and neutrophil elastase.

Proteases play a critical role in the ordered remodelling of extracellular matrix (ECM) components during wound healing and tissue regeneration. However, the usually ordered proteolysis is compromised in chronic wounds due to over-expression and high concentrations of matrix metalloproteinase's (MMPs) and neutrophil elastase (NE). Ovine forestomach matrix (OFM) is a decellularised extracellular matrix-based biomaterial developed for tissue regeneration applications, including the treatment of chronic wounds, and is a heterogeneous mixture of ECM proteins and proteoglycans that retains the native structural and functional characteristics of tissue ECM. Given the diverse molecular species present in OFM, we hypothesised that OFM may contain components or fragments that inhibit MMP and NE activity. An extract of OFM was shown to be a potent inhibitor of a range of tissue MMPs (IC50 s = 23 ± 5 to 115 ± 14 µg/ml) and NE (IC50 = 157 ± 37 µg/ml), and was more potent than extracts prepared from a known protease modulating wound dressing. The broad spectrum activity of OFM against different classes of MMPs (i.e. collagenases, gelatinases and stromelysins) may provide a clinical advantage by more effectively addressing the protease imbalance seen in chronic wounds.

Clinical outcomes following the use of ovine forestomach matrix (endoform dermal template) to treat chronic wounds.

The suitability of the ovine forestomach matrix (OFM) for the treatment of recalcitrant wounds was evaluated in 19 patients. At 12 weeks, 50% of wounds had closed, and the average reduction in surface area was 73.4%. Promising outcomes of this initial series support the clinical consideration of OFM.

Ovine forestomach matrix as a substrate for single-stage split-thickness graft reconstruction.

OBJECTIVE: Split skin graft reconstruction of scalp defects often leaves an obvious contour defect. Here, we aimed to demonstrate the use of a decellularized extracellular matrix biomaterial, termed ovine forestomach matrix (OFM), as a substrate for split-thickness skin grafts (STSGs) for scalp reconstruction. METHODS: Following full-thickness tumor excision, OFM was applied directly to skull periosteum, and then an STSG was applied. Participants were monitored for graft take, epithelialization, and cosmetic outcomes. RESULTS: Participants responded well to the procedure with more than 95% graft take in 4 participants, and 100% epithelialization of the grafts after 2 weeks. A 30% graft take was observed in the fifth participant due to local infection and partial necrosis of the graft. Ovine forestomach matrix was remodelled with time and the regenerated dermis was well vascularized and had robust and ordered collagen deposition. CONCLUSIONS: This series demonstrates that OFM can serve as a temporary dermal scaffold to support an overlying STSG and allow for a single-stage grafting procedure.

Pharmacokinetics of quinacrine efflux from mouse brain via the P-glycoprotein efflux transporter.

The lipophilic cationic compound quinacrine has been used as an antimalarial drug for over 75 years but its pharmacokinetic profile is limited. Here, we report on the pharmacokinetic properties of quinacrine in mice. Following an oral dose of 40 mg/kg/day for 30 days, quinacrine concentration in the brain of wild-type mice was maintained at a concentration of ∼1 µM. As a substrate of the P-glycoprotein (P-gp) efflux transporter, quinacrine is actively exported from the brain, preventing its accumulation to levels that may show efficacy in some disease models. In the brains of P-gp-deficient Mdr1(0/0) mice, we found quinacrine reached concentrations of ∼80 µM without any signs of acute toxicity. Additionally, we examined the distribution and metabolism of quinacrine in the wild-type and Mdr1(0/0) brains. In wild-type mice, the co-administration of cyclosporin A, a known P-gp inhibitor, resulted in a 6-fold increase in the accumulation of quinacrine in the brain. Our findings argue that the inhibition of the P-gp efflux transporter should improve the poor pharmacokinetic properties of quinacrine in the CNS.

Quantification of in vitro and in vivo angiogenesis stimulated by ovine forestomach matrix biomaterial.

Ovine forestomach matrix (OFM) biomaterial acts as a biomimetic of native extracellular matrix (ECM) by providing structural and functional cues to orchestrate cell activity during tissue regeneration. The ordered collagen matrix of the biomaterial is supplemented with secondary ECM-associated macromolecules that function in cell adhesion, migration and communication. As angiogenesis and vasculogenesis are critical processes during tissue regeneration we sought to quantify the angiogenic properties of the OFM biomaterial. In vitro studies demonstrated that soluble OFM components stimulated human umbilical vein endothelial cell (HUVEC) migration and increased vascular sprouting from an aorta. Blood vessel density and branch points increased in response to OFM in an ex ovo chicken chorioallantoic membrane (CAM) assay. The OFM biomaterial was shown to undergo remodeling in a porcine full-thickness excisional model and gave rise to significantly more blood vessels than wounds treated with small intestinal submucosa decellularized ECM or untreated wounds.

Biophysical characterization of ovine forestomach extracellular matrix biomaterials.

Ovine forestomach matrix (OFM) is a native and functional decellularized extracellular matrix biomaterial that supports cell adhesion and proliferation and is remodeled during the course of tissue regeneration. Small angle X-ray scattering demonstrated that OFM retains a native collagen architecture (d spacing = 63.5 ± 0.2 nm, orientation index = 20°). The biophysical properties of OFM were further defined using ball-burst, uniaxial and suture retention testing, as well as a quantification of aqueous permeability. OFM biomaterial was relatively strong (yield stress = 10.15 ± 1.81 MPa) and elastic (modulus = 0.044 ± 0.009 GPa). Lamination was used to generate new OFM-based biomaterials with a range of biophysical properties. The resultant multi-ply OFM biomaterials had suitable biophysical characteristics for clinical applications where the grafted biomaterial is under load.

A functional extracellular matrix biomaterial derived from ovine forestomach.

Extracellular matrix (ECM) based biomaterials have an established place as medical devices for wound healing and tissue regeneration. In the search for biomaterials we have identified ovine forestomach matrix (OFM), a thick, large format ECM which is biochemically diverse and biologically functional. OFM was purified using an osmotic process that was shown to reduce the cellularity of the ECM and aid tissue delamination. OFM produced using this technique was shown to retain residual basement membrane components, as evidence by the presence of laminin and collagen IV. The collagenous microarchitecture of OFM retained many components of native ECM including fibronectin, glycosaminoglycans, elastin and fibroblast growth factor basic. OFM was non-toxic to mammalian cells and supported fibroblast and keratinocyte migration, differentiation and infiltration. OFM is a culturally acceptable alternative to current collagen-based biomaterials and has immediate clinical applications in wound healing and tissue regeneration.

Discovery of 2-aminothiazoles as potent antiprion compounds.

Prion diseases are fatal, untreatable neurodegenerative diseases caused by the accumulation of the misfolded, infectious isoform of the prion protein (PrP), termed PrP(Sc). In an effort to identify novel inhibitors of prion formation, we utilized a high-throughput enzyme-linked immunosorbent assay (ELISA) to evaluate PrP(Sc) reduction in prion-infected neuroblastoma cell lines (ScN2a). We screened a library of approximately 10,000 diverse small molecules in 96-well format and identified 121 compounds that reduced PrP(Sc) levels at a concentration of 5 microM. Four chemical scaffolds were identified as potential candidates for chemical optimization based on the presence of preliminary structure-activity relationships (SAR) derived from the primary screening data. A follow-up analysis of a group of commercially available 2-aminothiazoles showed this class as generally active in ScN2a cells. Our results establish 2-aminothiazoles as promising candidates for efficacy studies of animals and validate our drug discovery platform as a viable strategy for the identification of novel lead compounds with antiprion properties.

Molecular modeling of the misfolded insulin subunit and amyloid fibril.

Insulin, a small hormone protein comprising 51 residues in two disulfide-linked polypeptide chains, adopts a predominantly alpha-helical conformation in its native state. It readily undergoes protein misfolding and aggregates into amyloid fibrils under a variety of conditions. Insulin is a unique model system in which to study protein fibrillization, since its three disulfide bridges are retained in the fibrillar state and thus limit the conformational space available to the polypeptide chains during misfolding and fibrillization. Taking into account this unique conformational restriction, we modeled possible monomeric subunits of the insulin amyloid fibrils using beta-solenoid folds, namely, the beta-helix and beta-roll. Both models agreed with currently available biophysical data. We performed molecular dynamics simulations, which allowed some limited insights into the relative structural stability, suggesting that the beta-roll subunit model may be more stable than the beta-helix subunit model. We also constructed beta-solenoid-based insulin fibril models and conducted fiber diffraction simulation to identify plausible fibril architectures of insulin amyloid. A comparison of simulated fiber diffraction patterns of the fibril models to the experimental insulin x-ray fiber diffraction data suggests that the model fibers composed of six twisted beta-roll protofilaments provide the most reasonable fit to available experimental diffraction patterns and previous biophysical studies.

Site-directed mutagenesis demonstrates the plasticity of the beta helix: implications for the structure of the misfolded prion protein.

The left-handed parallel beta helix (LbetaH) fold has recently received attention as a possible structure for the prion protein (PrP) in its misfolded state. In light of this interest, we have developed an experimental system to examine the structural requirements of the LbetaH fold, using a known LbetaH protein, UDP-N-acetylglucosamine acyltransferase (LpxA), from E. coli. We showed that the beta helix can tolerate nonhydrophobic residues at interior positions and prolines were important, but not critical, in folding of the beta helix. Using our structural studies of the LbetaH, we threaded the sequence of the amyloidogenic fragment of the prion protein (residues 104-143) onto the structure of LpxA. Based on the threading result, we constructed the recombinant PrP-LpxA and tested its functional activity in an E. coli antibiotic sensitivity assay. The results of these experiments suggest that the amyloidogenic PrP fragment may fold into a beta helix in the context of a larger beta-helical structure.