Formulation and characterization of gelatin methacrylamide-hydroxypropyl methacrylate based bioink for bioprinting applications.

Three-dimensional (3D) bioprinting has emerged as a revolutionary technology for constructing functional tissue equivalents/scaffolds for tissue engineering applications. Bioink design is a crucial element in 3D bioprinting, which typically comprises a mixture of biomaterials, biological molecules or cells followed by its printing and tissue maturation. An ideal bioink should possess suitable physicochemical, mechanical, rheological, and biological features of the target tissue. However, mimicking multifaceted compositions similar to native extracellular matrix (ECM) with bioactive milieu of soluble and non-soluble factors is challenging. Herein, we report the formulation and characterization of a bioink system, comprising methacrylamide modified gelatin (GelMA) and 2-hydroxylpropyl methacrylate (HPMA) with a cost-effective redox initiators based cross-linking. GelMA was synthesized by reacting gelatin with methacrylic anhydride (MA) and subsequently, copolymerized with HPMA at room temperature by redox mechanism. Various hydrogel formulations by varying GelMA: HPMA w/v% ratios (G:HP) were studied as 10:0 (G100HP0), 9.5:0.5 (G95HP05), 9:1 (G90HP10), 8:2 (G80HP20), and 6:4 (G60HP40), to identify the best bioink composition. The formulations were characterized for its opacity, chemical, rheological, mechanical, porosity and swelling properties and cytocompatibility as per ISO-10993 standards. Cell encapsulation studies using live/dead assay analyzed cell viability inside the handprinted and 3D printed constructs. The preliminary results indicate successful formulation of cytocompatible bioink for potential 3D bioprinting and biofabrication applications.

Assembly of skin substitute by cross-linking natural biomaterials on synthetic biodegradable porous mat for critical-size full-thickness burn wound regeneration.

Human skin architecture comprises several interpenetrating macromolecules seen as organized extracellular matrix (ECM). For regeneration of critical-size acute and chronic wounds, substituting the damaged tissue with artificially assembled biomolecules offer an interactivemilieu. This study reports development and preclinical evaluation of a biodegradable and immuno-compatible scaffold for regeneration of critical-size (4 × 4 cm2) full-thickness rabbit burn wounds. The designed wound care product comprises synthetic terpolymer poly(L-Lactide-co-Glycolide-co-Caprolactone) (PLGC), human clinical-grade fibrin (FIB), and hyaluronic acid (HA), termed as PLGCFIBHA. Here, clotting of fibrinogen concentrate (FC) with excess thrombin in the scaffold create an interpenetrating FIB network harnessed with adhesive molecules like fibronectin and laminin present in FC with exogenous HA to produce ECM-likemilieuon porous PLGC. Penetrating into porous PLGCFIBHA, long term study showed a regulated fibroblast growth resulting in non-fibrotic dermal-like tissuein vitro. The freeze-dried PLGCFIBHA with residual thrombin facilitated suture-less, hemostatic matrix adhesion to the wound bedin vivo. By 28 d, mature and scar-less epidermis-dermis formation with skin appendages was evident in the PLGCFIBHA-treated wound area. Both negative (untreated/sham) and positive (commercial matrix-treated) control wounds showed incomplete regeneration. The PLGCFIBHA-treated wounds were comparable to native skin by 56 d. These regenerative outcomes upon single application of PLGCFIBHA confirms its potential translational value for wound care.

Extracellular matrix-based combination scaffold for guided regeneration of large-area full-thickness rabbit burn wounds upon a single application.

Regeneration of large acute and chronic wounds is a concern worldwide. The present study evaluates wound healing competence of a completely human-origin, extracellular matrix (ECM)-based skin substitute/graft. It comprises cell-less amniotic membrane (AM), clinical-grade fibrin (FIB), and hyaluronic acid (HA) termed as AMFIBHA. The use of large-area third-degree rabbit burn wounds evaluated the product efficiency. The AMFIBHA induces hemostasis and permits suture-less positioning on the wound bed. In wet wounds, the AMFIBHA degrades and release biologically active molecules and guide cell migration, proliferation, and regeneration. The study demonstrated the effectiveness of this wound care product in terms of epithelial-dermal regeneration with angiogenesis. The study assessed injury-associated inflammation and different wound healing markers after 28 days of experiment and compared with both positive and negative controls-treated wounds. The regeneration of mature epidermis and dermis with rete pegs and hair follicle-like structure was evident upon a single application. The active involvement of host cells resulted in supple tissue formation. The ECM organization of AMFIBHA-treated tissue resulted in re-gain of mechanical properties comparable to native skin after 56 days. These guided regenerative outcomes reveal a promising translational value of the novel AMFIBHA skin substitute as an off-the-shelf product for clinical use.

Silk Fibroin-Based Bioengineered Scaffold for Enabling Hemostasis and Skin Regeneration of Critical-Size Full-Thickness Heat-Induced Burn Wounds.

Millions of people around the globe are affected by full-thickness skin injuries. A delay in the healing of such injuries can lead to the formation of chronic wounds, posing several clinical and economic challenges. Current strategies for wound care aim for skin regeneration and not merely skin repair or faster wound closure. The present study aimed to develop a bioactive wound-healing matrix comprising natural biomaterial silk fibroin (SF), clinical-grade human fibrin (FIB), and human hyaluronic acid (HA), resulting in SFFIBHA for regeneration of full-thickness burn wounds. A porous, hemostatic, self-adhesive, moisture-retentive, and biomimetic scaffold that promotes healing was the expected outcome. The study validated a terminal sterilization method, suggesting the stability and translational potential of the novel scaffold. Also, the study demonstrated the regenerative abilities of scaffolds using in vitro cell culture experiments and in vivo full-thickness burn wounds of critical size (4 cm × 4 cm) in a rabbit model. Under in vitro conditions, the scaffold enhanced primary dermal fibroblast adhesion and cell proliferation with regulated extracellular matrix (ECM) synthesis. In vivo, the scaffolds promoted healing with mature epithelium coverage involving intact basal cells, superficial keratinocytes, multilayers of keratohyalin, dermal regeneration with angiogenesis, and deposition of remodeled ECM in 28 days. The relative gene expression of the IL6 marker indicated transitions from inflammation to proliferation stage. In addition, we observed skin appendages and rete peg development in the SFFIBHA-treated wound tissues. Although wound closure was observed, neither negative (untreated/sham) nor positive (commercially available product; NeuSkin) control wounds developed skin appendages/rete pegs or native skin architecture. After 56 days, healing with organized ECM production enabled the recovery of mechanical properties of skin with higher tissue maturity in SFFIBHA-treated wounds. Thus, in a single application, the SFFIBHA scaffold proved to be an efficient biomimetic matrix that can guide burn wound regeneration. The developed matrix is a suture-less, hemostatic, off-the-shelf product for potential wound regenerative applications.

Generation of niche tuned antifibrotic fibroblasts and non-viral mediated endothelial commitment using adipose stem cells for dermal graft development.

Cell-based skin substitute generation has seen considerable development. Combining synthetic scaffolds with biomimetic fibrin does direct both exogenous and endogenous stem cell differentiation, addressing needs for reliable tissue engineering. However, lack of immediate vasculature within implantable grafts remains critical for its sustenance and integration. Multipotency, high proliferation potential, ability to release multiple growth factors (GFs), and autologous availability highlight the use of human adipose derived mesenchymal stem cells (hADMSCs) in tissue-engineered dermal grafts (TEDG) construction. However, hADMSCs' insufficiency to independently establish angiogenesis within tissue constructs demands improvement of stem cell application for dermal graft survival. Approaches to harness microenvironmentally sensitive paracrine interactions could improve the angiogenic efficiency of hADMSCs within TEDG. This study conceptualized a fibrin-based niche, to direct hADMSCs toward a nonfibrotic fibroblast commitment and incorporation of bioengineered hADMSCs, specifically releasing potent angiogenic factors within TEDG. Coexistence of tuned fibroblast and endothelial lineage committed cells contributed to well-regulated extracellular matrix formation and prevascularization. Adequate cell proliferation; sustained transient release of angiogenic GFs till 20 days; directed dermal, endothelial, fibroblast, and vascular smooth muscle cell differentiation; and favored elastin and collagen deposition were achieved in vitro. In conclusion, specific niche composition and employment of bioengineered hADMSCs favor implantable TEDG construction.

Human-Derived Scaffold Components and Stem Cells Creating Immunocompatible Dermal Tissue Ensuing Regulated Nonfibrotic Cellular Phenotypes.

Regeneration of large-sized acute and chronic wounds provoked by severe burns and diabetes is a major concern worldwide. The availability of immunocompatible matrix with a wide range of regenerative medical applications, more specifically, for nonhealing chronic wounds is an unmet clinical need. Extrapolating the in vitro tissue engineering knowledge for in vivo guided wound regeneration could be a meaningful approach. This study aimed to develop a completely human-derived and minimally immune-responsive scaffold comprising of acellular amniotic membrane (AM), fibrin (FIB) and hyaluronic acid (HA), termed AMFIBHA. The potential for in vivo guidance of skin regeneration was validated through in vitro dermal tissue assembly on the combination scaffold by growing human fibroblasts, differentiated from human adipose tissue-derived mesenchymal stem cells (hADMSCs). An effective method was standardized for obtaining decellularized amnion (dAM) for assuring better immuno-compatibility. The biochemical stability of dAM upon plasma sterilization (pdAM) confirms its suitability for both in vitro and in vivo tissue engineering. The problem of poor handling characteristics was solved by combining the dried dAM with fibrin derived from a clinically used fibrin sealant kit. An additional constituent HA, derived from human umbilical cord tissue, imparts the required water absorption and retention property for better cell migration and growth. Post sterilization, the combination scaffold AMFIBHA demonstrated hemo-/cytocompatibility, confirming the absence of detergent residuals. Upon long-term (20 days/40 days) culture of hADMSC-derived fibroblasts, the suppleness of generated tissue was established by demonstrating regulated deposition of collagen, elastin, and glycosaminoglycans using both qualitative and quantitative measurements. Regulated expressions of transforming growth factors-beta 1 (TGF-ß1) & TGF-ß3, alpha smooth muscle actin (α-SMA), fibrillin-1, collagen subtypes, and elastin suggest non-fibrotic fibroblast phenotype, which could be an effect of microenvironment endowed by the AM, FIB, and HA. In burn wound model experiments, immune response to cellular AM was prominent as compared to untreated/sham control wounds and decellularized AM-treated and AMFIBHA-treated wounds, ensuring biocompatibility. Wound regeneration with complete epithelialization, angiogenesis, development of rete pegs, and other skin appendages were clearly visualized in 28 days after treating large-sized (4 × 4 cm2), debrided, full-thickness third-degree burn wounds, indicating guided wound regeneration potential of AMFIBHA dermal substitute.