3D Bioprinting Skin Applications, Wound Healing

Category: Blog,From Academia
Dec 26, 2020

3D bioprinting skin applications including wound healing have been a focus of key biofabrication development. In this issue of “From Academia”, we included three publications studying just that. The first study develops a bioprinted skin patch with antimicrobial and wound healing properties using 3D bioprinting. The second study demonstrated improved physical and biological characteristics of fibrinogen hydrogel supplemented with decellularized human skin-derived extracellular matrix (dsECM). This hybrid hydrogel improves the cell viability and structural strength of bioprinted skin constructs, hence better-wound healing ability. The final study develops 3D-printed biomimetic wound dressings using melt eletronwriting technique and demonstrates a solution that could potentially reduce scar tissue formation while enhancing wound closure. From Academia” features recent, relevant, close to commercialization academic publications. Subjects include but not limited to healthcare 3D printing, 3D bioprinting, and related emerging technologies.

Email: Rance Tino ([email protected]) if you want to share relevant academic publications with us.

Development of Bio-Active Patches Based on Pectin for the Treatment of Ulcers and Wounds Using 3D-Bioprinting Technology

Authored by Eleftherios G. Andriotis,Georgios K. Eleftheriadis,Christina Karavasili and Dimitrios G. Fatouros. MDPI Pharmaceutics. 6 January 2020

Abstract: 

Biodegradable 3D-printable inks based on pectin have been developed as a system for direct and indirect wound-dressing applications, suitable for 3D printing technologies. The 3D-printable inks formed free-standing transparent films upon drying, with the latter exhibiting fast disintegration upon contact with aqueous media. The antimicrobial and wound-healing activities of the inks have been successfully enhanced by the addition of particles, comprised of chitosan and cyclodextrin inclusion complexes with propolis extract. Response Surface Methodology (RSM) was applied for the optimization of the inks (extrusion-printing pressure, shrinkage minimization over-drying, increased water uptake and minimization of the disintegration of the dry patches upon contact with aqueous media). Particles comprised of chitosan and cyclodextrin/propolis extract inclusion complexes (CCP), bearing antimicrobial properties, were optimized and integrated with the produced inks. The bioprinted patches were assessed for their cytocompatibility, antimicrobial activity and in vitro wound-healing properties. These studies were complemented with ex vivo skin adhesion measurements, a relative surface hydrophobicity and opacity measurement, mechanical properties, visualization, and spectroscopic techniques. The in vitro wound-healing studies revealed that the 3D-bioprinted patches enhanced the in vitro wound-healing process, while the incorporation of CCP further enhanced wound-healing, as well as the antimicrobial activity of the patches.

Antimicrobial activity of 3D-printed pectin films with: 0%, 2.5%, 5%, 10%, 20% and 30% w/w CCP, against two bacterial strains of interest. *** P < 0.05 vs. control. Copyright MDPI Pharmaceutics
Antimicrobial activity of 3D-printed pectin films with: 0%, 2.5%, 5%, 10%, 20% and 30% w/w CCP, against two bacterial strains of interest. *** P < 0.05 vs. control. Copyright MDPI Pharmaceutics

Decellularized Skin Extracellular Matrix (dsECM) Improves the Physical and Biological Properties of Fibrinogen Hydrogel for Skin Bioprinting Applications

Authored by Adam M Jorgensen, Zishuai Chou, Gregory Gillispie, Sang Jin Lee, James J Yoo, Shay Soker and Anthony Atala. MDPI Nanomaterials. 24 July 2020 

Improved biological characteristics of fibrinogen hydrogel supplemented with decellularized human skin extracellular matrix (dsECM). (A) Masson’s trichrome stained human skin compared with bioprinted skin bioprinted with a fibrinogen hydrogel (scale bars, 50 µm). (B) A graphical model describing the matrix components of fibrinogen hydrogel, decellularized skin ECM (dsECM), fibrinogen hydrogel supplemented with dsECM, and normal human skin ECM (created with BioRender.com). (C) H&E stained molded skin constructs with fibrinogen hydrogel supplemented with dsECM vs. fibrinogen only over 15 days (scale bars, 50 µm). (D) Live/dead cell viability assay demonstrates that viability was better maintained at day 15 in the fibrinogen hydrogel supplemented with dsECM. (E) Rheological assessment demonstrates improved mechanical strength of fibrinogen hydrogel supplemented with dsECM over 15 days in culture, with increased mechanical strength in cellularized constructs compared with gel only constructs.. Copyright MDPI Nanomaterials
Improved biological characteristics of fibrinogen hydrogel supplemented with decellularized human skin extracellular matrix (dsECM). (A) Masson’s trichrome stained human skin compared with bioprinted skin bioprinted with a fibrinogen hydrogel (scale bars, 50 µm). (B) A graphical model describing the matrix components of fibrinogen hydrogel, decellularized skin ECM (dsECM), fibrinogen hydrogel supplemented with dsECM, and normal human skin ECM (created with BioRender.com). (C) H&E stained molded skin constructs with fibrinogen hydrogel supplemented with dsECM vs. fibrinogen only over 15 days (scale bars, 50 µm). (D) Live/dead cell viability assay demonstrates that viability was better maintained at day 15 in the fibrinogen hydrogel supplemented with dsECM. (E) Rheological assessment demonstrates improved mechanical strength of fibrinogen hydrogel supplemented with dsECM over 15 days in culture, with increased mechanical strength in cellularized constructs compared with gel only constructs.. Copyright MDPI Nanomaterials

Abstract: 

Full-thickness skin wounds are a significant clinical burden in the United States. Skin bioprinting is a relatively new technology that is under investigation as a new treatment for full-thickness injuries, and development of hydrogels with strong physical and biological characteristics are required to improve both structural integrity of the printed constructs while allowing for a more normal extracellular matrix milieu. This project aims to evaluate the physical and biological characteristics of fibrinogen hydrogel supplemented with decellularized human skin-derived extracellular matrix (dsECM). The hybrid hydrogel improves the cell viability and structural strength of bioprinted skin constructs. Scanning electron microscopy demonstrates that the hybrid hydrogel is composed of both swelling bundles interlocked in a fibrin network, similar to healthy human skin. This hybrid hydrogel has improved rheological properties and shear thinning properties. Extrusion-based printing of the fibrinogen hydrogel + dsECM demonstrates significant improvement in crosshatch pore size. These findings suggest that incorporating the properties of dsECM and fibrinogen hydrogels will improve in vivo integration of the bioprinted skin constructs and support of healthy skin wound regeneration. 

Improved physical characteristics of fibrinogen hydrogel supplemented with decellularized human skin extracellular matrix (dsECM). Rheological tests including (A) storage modulus (G’) temperature sweep, (B) viscosity over shear rate, and (C) viscosity temperature sweep demonstrate the shear-thinning properties and theoretical printability of the fibrinogen + dsECM hydrogel. (D) Storage modulus (G’) pre and post-crosslinking with thrombin. (E) Scanning electron microscopy (SEM) of each gel type compared to decellularized human skin; small fibrils (arrows, →) and swelling bundles (stars, ☆). Copyright MDPI Nanomaterials
Improved physical characteristics of fibrinogen hydrogel supplemented with decellularized human skin extracellular matrix (dsECM). Rheological tests including (A) storage modulus (G’) temperature sweep, (B) viscosity over shear rate, and (C) viscosity temperature sweep demonstrate the shear-thinning properties and theoretical printability of the fibrinogen + dsECM hydrogel. (D) Storage modulus (G’) pre and post-crosslinking with thrombin. (E) Scanning electron microscopy (SEM) of each gel type compared to decellularized human skin; small fibrils (arrows, →) and swelling bundles (stars, ☆). Copyright MDPI Nanomaterials

Convergence of 3D printed biomimetic wound dressings and adult stem cell therapy

Authored by Abbas Shafiee, Amanda S. Cavalcanti, navid T. Saidy, Dominik Schneidereit, Oliver Friedrich, Akhilandeshwari Ravichandran, Elena M De-Juan-Pardo, Dietmar W. Hutmacher. Biomaterials. January 2021

Schematic illustration of fabrication, plasma treatment (A) and cell seeding (B) on the melt electro written (MEW) anisotropic medical-grade polycaprolactone (mPCL) dressings. C) Two full-thickness skin wounds were created in the dorsal skin in a rat model using a disposable 10.0 mm biopsy punch tool. D) Wounds were stented with custom-made stainless-steel splints (15 mm in diameter) and then mPCL alone or mPCL seeded with human gingival tissue derived multipotent mesenchymal stem/stromal cells (MSCs) were transplanted into the wounds. E) On day 7, the splints were removed, and the biomimetic dressing was spontaneously ejected from the healed wound bed in week 2. The regenerated wounds were analyzed at the experimental endpoint, day 42. Copyright. Biomaterials
Schematic illustration of fabrication, plasma treatment (A) and cell seeding (B) on the melt electro written (MEW) anisotropic medical-grade polycaprolactone (mPCL) dressings. C) Two full-thickness skin wounds were created in the dorsal skin in a rat model using a disposable 10.0 mm biopsy punch tool. D) Wounds were stented with custom-made stainless-steel splints (15 mm in diameter) and then mPCL alone or mPCL seeded with human gingival tissue derived multipotent mesenchymal stem/stromal cells (MSCs) were transplanted into the wounds. E) On day 7, the splints were removed, and the biomimetic dressing was spontaneously ejected from the healed wound bed in week 2. The regenerated wounds were analyzed at the experimental endpoint, day 42. Copyright. Biomaterials

Abstract: 

Biomimetically designed medical-grade polycaprolactone (mPCL) dressings are 3D-printed with pore architecture and anisotropic mechanical characteristics that favor skin wound healing with reduced scarring. Melt electrowritten mPCL dressings are seeded with human gingival tissue multipotent mesenchymal stem/stromal cells and cryopreserved using a clinically approved method. The regenerative potential of fresh or frozen cell-seeded mPCL dressing is compared in a splinted full-thickness excisional wound in a rat model over six weeks. The application of 3D-printed mPCL dressings decreased wound contracture and significantly improved skin regeneration through granulation and re-epithelialization compared to control groups. Combining 3D-printed biomimetic wound dressings and precursor cell delivery enhances physiological wound closure with reduced scar tissue formation.

HGMSC-seeded dressing displays the best wound healing potential with the least scar area. A) The absence of human cells in the wound site after implantation of wound dressings was confirmed using immunohistochemical (IHC) staining for human-specific Lamin A/C antibody, representative images of frozen tissue engineered constructs (TEC, cell seeded dressing), and human breast cancer xenograft (positive control), scale bar: 200 μm. B) IHC staining for CD31 showed the wound beds were vascularized and similar levels of CD31+ area were observed among all the experimental groups, scale bar: 200 μm. C) The cell-seeded dressing accelerated the full-thickness dermal wound healing and displayed the least scar area and the most differentiated epithelium among other groups with the most similarity to the native skin. Abbreviation: FreshTEC: fresh tissue-engineered constructs (fresh cell-seeded dressings), CroyoTEC: cryopreserved tissue-engineered constructs (cryopreserved cell-seeded dressings). HGMSC: Human gingiva derived multipotent mesenchymal stem/stromal cells. Copyright. Biomaterials
HGMSC-seeded dressing displays the best wound healing potential with the least scar area. A) The absence of human cells in the wound site after implantation of wound dressings was confirmed using immunohistochemical (IHC) staining for human-specific Lamin A/C antibody, representative images of frozen tissue engineered constructs (TEC, cell seeded dressing), and human breast cancer xenograft (positive control), scale bar: 200 μm. B) IHC staining for CD31 showed the wound beds were vascularized and similar levels of CD31+ area were observed among all the experimental groups, scale bar: 200 μm. C) The cell-seeded dressing accelerated the full-thickness dermal wound healing and displayed the least scar area and the most differentiated epithelium among other groups with the most similarity to the native skin. Abbreviation: FreshTEC: fresh tissue-engineered constructs (fresh cell-seeded dressings), CroyoTEC: cryopreserved tissue-engineered constructs (cryopreserved cell-seeded dressings). HGMSC: Human gingiva derived multipotent mesenchymal stem/stromal cells. Copyright. Biomaterials

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