Tailored Extracellular Matrix-Biomimetic Coating Favors Tissue Healing by Modulating the Microenvironment.

Tailored extracellular matrix-mimetic coating facilitates reendothelialization and tissue healing of cardiac occluders

PurposeTo determine feasibility of plant-derived recombinant human collagen type I (RHCI) for use in corneal regenerative implantsMethodsRHCI was crosslinked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to form hydrogels. Application of shear force to liquid crystalline RHCI aligned the collagen fibrils. Both aligned and random hydrogels were evaluated for mechanical and optical properties, as well as in vitro biocompatibility. Further evaluation was performed in vivo by subcutaneous implantation in rats and corneal implantation in Göttingen minipigs.ResultsSpontaneous crosslinking of randomly aligned RHCI (rRHCI) formed robust, transparent hydrogels that were sufficient for implantation. Aligning the RHCI (aRHCI) resulted in thicker collagen fibrils forming an opaque hydrogel with insufficient transverse mechanical strength for surgical manipulation. rRHCI showed minimal inflammation when implanted subcutaneously in rats. The corneal implants in minipigs showed that rRHCI hydrogels promoted regeneration of corneal epithelium, stroma, and nerves; some myofibroblasts were seen in the regenerated neo-corneas.ConclusionPlant-derived RHCI was used to fabricate a hydrogel that is transparent, mechanically stable, and biocompatible when grafted as corneal implants in minipigs. Plant-derived collagen is determined to be a safe alternative to allografts, animal collagens, or yeast-derived recombinant human collagen for tissue engineering applications. The main advantage is that unlike donor corneas or yeast-produced collagen, the RHCI supply is potentially unlimited due to the high yields of this production method.Lay SummaryA severe shortage of human-donor corneas for transplantation has led scientists to develop synthetic alternatives. Here, recombinant human collagen type I made of tobacco plants through genetic engineering was tested for use in making corneal implants. We made strong, transparent hydrogels that were tested by implanting subcutaneously in rats and in the corneas of minipigs. We showed that the plant collagen was biocompatible and was able to stably regenerate the corneas of minipigs comparable to yeast-produced recombinant collagen that we previously tested in clinical trials. The advantage of the plant collagen is that the supply is potentially limitless.

Hypertrophic scar development is a complication associated with wound healing, impacting local appearance and function. The type I/III collagen ratio affects the extent of hypertrophic scarring; a reduced ratio can ameliorate this. In this study, recombinant human collagen type III was developed. Liquid chromatography-tandem mass spectrometry was used to determine its amino acid sequence and confirm its high level of homology with natural human type III collagen. Recombinant human collagen type III displayed no cytotoxicity and did not confer skin irritation and sensitization. Immunofluorescence and western blot analyses of histidine following incubation with fibroblasts suggested cell entry of recombinant human collagen type III. Furthermore, recombinant human collagen type III promoted the synthesis of the natural type III collagen in fibroblasts, resulting in a more obvious increase of type III collagen content in fibroblasts than that of type I collagen, and then decreased the ratio of type I/III collagen. The results of 5-ethynyl-2'-deoxyuridine staining assay suggested enhanced fibroblast proliferation. Following local injection of recombinant human collagen type III, rabbit ear scarring was significantly reduced after 60 days. Vancouver Scar Scale evaluation showed that all index scores were significantly reduced. Western blotting and Picro-Sirius red staining showed that the natural type III collagen increase in scar tissue was greater than that of type I collagen, decreasing the type I/III ratio. In summary, recombinant human collagen type III can be taken up by fibroblasts and promote natural collagen synthesis-especially that of type III-thereby reducing the type I/III ratio and improving hypertrophic scarring.

Conducting tissue healing and regeneration through biomaterials and morphogens is still an unrealized goal. Understand the multiple roles of the extracellular matrix (ECM) is indeed essential for the design of successful regenerative medicine strategies. During tissue repair and healing, cells receive numerous signals from their immediate ECM microenvironment and adhere by receptor-mediated interactions with ECM components by specialized adhesion receptors, such as integrins. As such, design and modulation of ECM analogs to ligate specific integrins is a promising approach to control cellular processes. Through production of variants of the 9th to 10th type III repeat of fibronectin (FN, FN III9-10) with variable stabilities, we engineered ligands that present different specificities for the integrin α5β1. Furthermore, the FN fragments have been engineered in order to be covalently incorporated into fibrin, a clinical relevant matrix for regenerative medicine. We demonstrated the capacity of α5β1 integrin-specific engagement to influence human mesenchymal stem cell behavior, showing that α5β1 integrin has an important role in the control of their osteogenic differentiation. Specifically, compared to FN, FN fragments with increased specificity for α5β1 versus αvβ3 integrins (FN III9*-10) results in significantly enhanced osteogenic differentiation in 2D and in a clinically relevant fibrin matrix system, while cell attachment/spreading and proliferation were comparable. On the other hand, growth factors (GFs) are key molecules for tissue morphogenesis and healing. However, while they are really promising molecules for a use in regenerative medicine applications, they often fail to prove cost-effective or even clinically efficacious during clinical trials. One of the reasons for this poor translation may lie in the rapid clearance of GFs from tissue sites in vivo, leading to the development of strategies controlling their release. Since FN has been shown to bind GFs from very different families, we first explored the possibility of FN to bind GFs much more broadly, and secondly the possibility of using FN fragments as an anchor for GF retention into fibrin matrix. We found that the 12th to 14th type III repeats of FN (FN III12-14) promiscuously bind GFs from the platelet-derived GF, fibroblast GF, transforming GF-β and neurotrophin families. Overall, 25 new binding interactions were demonstrated, supporting that GF binding may be one of FN's main physiological functions. However, the reasons for such promiscuous binding capacity were still unclear, while evidences from the literature suggested that the close proximity of the major integrin-binding domain, FN III9-10, allows joint integrin/GF-receptor signaling triggered by a complex FN/GFs. Accordingly, we found that FN fragments containing both the integrin- and GF-binding domains (FN III9(*)-10/12-14) could drastically enhance GF activities in vitro. In addition, testing which integrins were involved within these synergistic effects, we found that α5β1 integrin was mainly involved. By the use of FN III9(*)-10/12-14 and fibrin, we could engineer a specific microenvironment allowing sequestration of multiple wild-type GFs, while triggering synergistic signaling between GF-receptors and integrins. In a delayed wound healing model in mouse and in a calvarial bone defect model in rat, GFs delivered with the FN fragment microenvironment were drastically improved in their ability to induce tissue healing, even though a single low dose of GFs was used. Specifically, we established integrin/GF-receptor synergistic activities as a key parameter for GF translation into regenerative medicine treatments and demonstrate a method to exploit this phenomenon. This thesis highlights the absolutely critical role of the microenvironment in modulating signaling of GFs and in driving these molecules forward toward more widespread clinical use.

Covalently immobilized recombinant humanized collagen type III (rhCol III) for enhanced anticoagulation and endothelialization.

Cutaneous wound healing is a complex process that strives to re-establish the original structure and functions of the skin. With the development of electrospinning technology, nanofibrous membrane biomaterials have emerged as promising pro-regenerative strategies for recapitulating the structure and composition of the natural extracellular matrix (ECM). Herein, a nanofibrous membrane wound dressing material based on recombinant human collagen type III (rhCol III) crosslinked by EDC/NHS (rhCol III EN NF), which incorporated multiple Gly-Glu-Lys (GEK) and Gly-Leu-Ser-Gly-Glu-Arg (GLSGER) integrin receptors, has been developed utilizing green electrospinning technology. The rhCol III EN NF exhibited excellent flexibility, mechanical properties and water absorption. Amino acid analysis showed that rhCol III EN NF retained integrin receptor-associated amino acids to mediate cell activities and then expedite wound healing. Subsequent in vitro experiments confirmed that the rhCol III EN NF effectively promotes cell adhesion, proliferation and migration. On a mouse full-thickness wound model, rhCol III EN NF dressings expedited wound closure and greatly improved collagen deposition, recovering dermal and epidermal structures as well as skin appendages. Altogether, our research demonstrated that rhCol III EN NF prepared by electrospinning technology could efficiently heal wounds and regenerate skin.

Background: In vitro three-dimensional (3D) hepatic spheroid culture has shown great promise in toxicity testing because it better mimics the cell–cell and cell–matrix interactions found in in vivo conditions than that of the traditional two-dimensional (2D) culture. Despite embedding HepaRG spheroids with collagen type I (collagen I) extracellular matrix (ECM) revealed a much better differentiation capability, almost all the collagen utilized in in vitro hepatocytes cultures is animal-derived collagen that may limit its use in human toxicity testing. Method: Here, a preliminary investigation of HepaRG cells cultured in different dimensionalities and with the addition of ECM was performed. Comparisons of conventional 2D culture with 3D spheroid culture were performed based on their functional or structural differences over 7 days. Rat tail collagen (rtCollagen) I and recombinant human collagen (rhCollagen) I were investigated for their ability in promoting HepaRG spheroid differentiation. Results: An immunofluorescence analysis of the hepatocyte-specific functional protein albumin suggested that HepaRG spheroids demonstrated better hepatic function than spheroids from 2D culture, and the function of HepaRG spheroids improved in a time-dependent manner. The fluorescence intensities per unit area of spheroids formed by 1000 cells on days 7 and 10 were 25.41 and 45.38, respectively, whereas almost undetectable fluorescence was obtained with 2D cells. In addition, the embedding of HepaRG spheroids into rtCollagen and rhCollagen I showed that HepaRG differentiation can be accelerated relative to the differentiation of spheroids grown in suspension, demonstrating the great promise of HepaRG spheroids. Conclusions: The culture conditions established in this study provide a potentially novel alternative for promoting the differentiation of HepaRG spheroids into mature hepatocytes through a collagen-embedded in vitro liver spheroid model. This culture method is envisioned to provide insights for future drug toxicology.

The innate immune response, particularly the phenotype of responding macrophages, has significant clinical implications in the remodeling outcome following implantation of biomaterials and engineered tissues. In general, facilitation of an anti-inflammatory (M2-like) phenotype is associated with tissue repair and favorable outcomes, whereas pro-inflammatory (M1-like) activation can contribute to chronic inflammation and a classic foreign body response. Biologic scaffolds composed of extracellular matrix (ECM) and, more recently, matrix-bound nanovesicles (MBV) embedded within the ECM are known to direct macrophages toward an anti-inflammatory phenotype and stimulate a constructive remodeling outcome. The mechanisms of MBV-mediated macrophage activation are not fully understood, but interleukin-33 (IL-33) within the MBV appears critical for M2-like activation. Previous work has shown that IL-33 is encapsulated within the lumen of MBV and stimulates phenotypical changes in macrophages independent of its canonical surface receptor stimulation-2 (ST2). In the present study, we used next-generation RNA sequencing to determine the gene signature of macrophages following exposure to MBV with and without intraluminal IL-33. MBV-associated IL-33 instructed an anti-inflammatory phenotype in both wild-type and st2-/- macrophages by upregulating M2-like and downregulating M1-like genes. The repertoire of genes regulated by ST2-independent IL-33 signaling were broadly related to the inflammatory response and crosstalk between cells of both the innate and adaptive immune systems. These results signify the importance of the MBV intraluminal protein IL-33 in stimulating a pro-remodeling M2-like phenotype in macrophages and provides guidance for the designing of next-generation biomaterials and tissue engineering strategies. Impact statement The phenotype of responding macrophages is predictive of the downstream remodeling response to an implanted biomaterial. The clinical impact of macrophage phenotype has motivated studies to investigate the factors that regulate macrophage activation. Matrix-bound nanovesicles (MBV) embedded within the extracellular matrix direct macrophages toward an anti-inflammatory (M2)-like phenotype that is indicative of a favorable remodeling response. Although the mechanisms of MBV-mediated macrophage activation are not fully understood, the intraluminal protein interleukin-33 (IL-33) is clearly a contributing signaling molecule. The present study identifies those genes regulated by MBV-associated IL-33 that promote a pro-remodeling M2-like macrophage activation state and can guide future therapies in regenerative medicine.

Non-selective (NSAIDs) and selective (COX-2) nonsteroidal anti-inflammatory drugs are commonly used for their analgesic and anti-inflammatory effects. Their role after orthopaedic surgery has been infrequently described and remains controversial because of unclear effects on soft tissue and bone healing. This study critically reviews the available literature to describe the effects of NSAIDs and COX-2 inhibitors on soft tissue and bone healing. A Medline search was performed using NSAIDs or COX-2 inhibitors and tissue healing. The combined search yielded 637 articles. Following exclusion, 44 articles were deemed relevant with 9 articles on soft tissue healing and 35 articles on bone healing. The available evidence is based primarily on animal data (39 studies), with considerable variation in study methods. In regard to soft tissue healing, there is insufficient evidence of a detrimental effect when using either NSAIDs or COX-2 inhibitors at standard doses for≤2weeks. For soft tissue to bone healing, a limited number of studies demonstrate impairment in healing. However, with respect to bone healing, indomethacin appears to have a clear detrimental effect, with less substantial evidence for other NSAIDs. Short-term, low-dose use of NSAIDs and COX-2 inhibitors does not appear to have a detrimental effect following soft tissue injury, but is inhibitory in cases involving bony healing. However, additional well-controlled human studies are necessary to draw more definitive conclusions regarding their role. Clinically, the prudent use of anti-inflammatory medications following sports medicine injuries and surgeries appears to be a reasonable option in clinical practice unless bone healing is required. III.

Biocompatibility control of recombinant collagen by ion beam modification

Methylglyoxal (MG) production after myocardial infarction (MI) leads to advanced glycation end‐product formation, adverse remodeling, and loss of cardiac function. The extracellular matrix (ECM) is a main target for MG glycation. This suggests that ECM‐mimicking biomaterial therapies may protect the post‐MI environment by removing MG. In this study, mechanisms by which a recombinant human collagen type I hydrogel therapy confers cardioprotection are investigated. One‐week post‐MI, mice receive intramyocardial injection of hydrogel or PBS. The hydrogel improves border zone contractility after 2 days, which is maintained for 28 days. RNA sequencing shows that hydrogel treatment decreases the expression of erythroid differentiation regulator 1, a factor associated with apoptosis. Hydrogel treatment reduces cardiomyocyte apoptosis and oxidative stress at 2 days with greater myocardial salvage seen at 28 days. The hydrogel located at the epicardial surface is modified by MG, and less MG‐modified proteins are observed in the underlying myocardium of hydrogel‐treated mice. Biomaterials that can be a target for MG glycation may act as a sponge to remove MG from the myocardium post‐MI. This leads to less oxidative stress, greater survival and contractility of cardiomyocytes, which altogether suggests a novel mechanism by which biomaterials improve function of the infarcted heart.

Prolyl 4-hydroxylase, the key enzyme of collagen synthesis, is an alpha2beta2 tetramer, the beta subunit of which is protein disulfide isomerase (PDI). Coexpression of the human alpha subunit and PDI in Pichia produced trace amounts of an active tetramer. A much higher, although still low, assembly level was obtained using a Saccharomyces pre-pro sequence in PDI. Coexpression with human type III procollagen unexpectedly increased the assembly level 10-fold, with no increase in the total amounts of the subunits. The recombinant enzyme was active not only in Pichia extracts but also inside the yeast cell, indicating that Pichia must have a system for transporting all the cosubstrates needed by the enzyme into the lumen of the endoplasmic reticulum. The 4-hydroxyproline-containing procollagen polypeptide chains were of full length and formed molecules with stable triple helices even though Pichia probably has no Hsp47-like protein. The data indicate that collagen synthesis in Pichia, and probably also in other cells, involves a highly unusual control mechanism, in that production of a stable prolyl 4-hydroxylase requires collagen expression while assembly of a stable collagen requires enzyme expression. This Pichia system seems ideal for the high-level production of various recombinant collagens for numerous scientific and medical purposes.

Injection of a recombinant human collagen hydrogel improves cardiac function and reduces pathological remodeling post myocardial infarction

Multi-omics elucidation of recombinant collagen-mediated modulation of mesenchymal stem cell functions.

Collagens carry out critical extracellular matrix (ECM) functions by interacting with numerous cell receptors and ECM components. Single glycine substitutions in collagen III, which predominates in vascular walls, result in vascular Ehlers-Danlos syndrome (vEDS), leading to arterial, uterine, and intestinal rupture and an average life expectancy of <50 years. Collagen interactions with integrin α2β1 are vital for platelet adhesion and activation; however, how these interactions are impacted by vEDS-associated mutations and by specific amino acid substitutions is unclear. Here, we designed collagen-mimetic peptides (CMPs) with previously reported Gly → Xaa (Xaa = Ala, Arg, or Val) vEDS substitutions within a high-affinity integrin α2β1-binding motif, GROGER. We used these peptides to investigate, at atomic-level resolution, how these amino acid substitutions affect the collagen III-integrin α2β1 interaction. Using a multitiered approach combining biological adhesion assays, CD, NMR, and molecular dynamics (MD) simulations, we found that these substitutions differentially impede human mesenchymal stem cell spreading and integrin α2-inserted (α2I) domain binding to the CMPs and were associated with triple-helix destabilization. Although an Ala substitution locally destabilized hydrogen bonding and enhanced mobility, it did not significantly reduce the CMP-integrin interactions. MD simulations suggested that bulkier Gly → Xaa substitutions differentially disrupt the CMP-α2I interaction. The Gly → Arg substitution destabilized CMP-α2I side-chain interactions, and the Gly → Val change broke the essential Mg2+ coordination. The relationship between the loss of functional binding and the type of vEDS substitution provides a foundation for developing potential therapies for managing collagen disorders.