RESUMEN
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.
Asunto(s)
Quemaduras , Piel Artificial , Animales , Materiales Biocompatibles , Quemaduras/terapia , Ácido Hialurónico , Porosidad , Conejos , Regeneración , Piel , Trombina , Cicatrización de HeridasRESUMEN
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.
Asunto(s)
Quemaduras , Piel Artificial , Animales , Quemaduras/terapia , Matriz Extracelular , Conejos , Piel , Trasplante de Piel , Cicatrización de HeridasRESUMEN
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.
Asunto(s)
Quemaduras , Piel Artificial , Colágeno , Humanos , Fenotipo , Cicatrización de HeridasRESUMEN
BACKGROUND AND AIM OF STUDY: Although the sheep is the most acceptable animal model for heart valve evaluation, it has severe limitations for detecting heart valve thrombosis during preclinical studies. While the pig offers an alternative model and is better for detecting prosthetic valve thrombogenicity, it is not often used because of inadvertent valve thrombosis or bleeding complications. The study aim was to develop an improved pig model which can be used reliably to evaluate mechanical heart valve thrombogenicity. METHODS: Mechanical heart valves were implanted in the mitral position of indigenous pigs administered aspirin-clopidogrel, and compared with similar valves implanted in control pigs to which no antiplatelet therapy had been administered. The pigs were observed for six months to study their overall survivability, inadvertent bleeding/valve thrombosis and pannus formation. The efficacy of aspirinclopidogrel on platelet aggregation and blood coagulation was also recorded and compared between test and control animals. RESULTS: In comparison to controls, pigs receiving anti-platelet therapy showed an overall better survivability, an absence of inadvertent valve thrombosis/ bleeding, and less obstructive pannus formation. Previously unreported inhibitory effects of aspirin-clopidogrel on the intrinsic pathway of blood coagulation were also observed in the pig model. Notably, with aspirin-clopidogrel therapy inadvertent thrombus formation or bleeding can be prevented. CONCLUSIONS: The newly developed pig model can be successfully used to evaluate heart valve thrombosis following chronic orthotopic valve implantation. The model may also be utilized to evaluate other bloodcontacting implantable devices.