ABSTRACT
Background: The availability of commercial tissue engineering skin repair products for veterinary use is scarce or non-existent. To assess features of novel veterinary tissue engineered medical devices, it is therefore reasonable to compare with currently available human devices. During the development and regulatory approval phases, human medical devices that may have been identified as comparable to a novel veterinary device, may serve as predicate devices and accelerate approval in the veterinary domain. The purpose of the study was to evaluate safety and efficacy of the biomembrane for use in skin repair indications. Methods: In the study as a whole (3 year total length), 15 patients (animals), dogs and cats (male/female, <8 years) with skin lesions of different etiologies considered difficult to heal (size, >2 cm), with a wound depth equivalent to 2nd/3rd degree burns are to be studied from Day 0 to Day 120-240, post-application of the biomembrane. This interim report covers the 5 patients assessed to date and deemed eligible, of which 3 enrolled, and 2 have completed the treatment. Wound beds were prepared and acellular collagen biomembranes (Eva Scientific Ltd, São Paulo, Brazil) applied directly onto the wounds, and sutured at the margins to the patient's adjacent tissue. Wound size over time, healing rate, general skin quality and suppleness were assessed as outcomes. Qualitative (appearance and palpation) and quantitative (based on Image Analysis of photographs) wound assessment techniques were used. Results: Both patients' wounds healed fully, with no adverse effects, and the healing rate was comparable in both, maxing out at approximately 1 cm 2/day. Conclusions: Early results on the biomembrane's safety and efficacy indicate suitability for skin repair usage in veterinary patients.
Subject(s)
Bandages , Burns/therapy , Collagen , Membranes, Artificial , Skin/injuries , Tissue Engineering , Wound Healing , Animals , Brazil , Burns/pathology , Cats , Collagen/chemistry , Collagen/pharmacology , Dogs , Female , Male , Skin/pathologyABSTRACT
INTRODUCTION: The optimization of an organic scaffold for specific types of applications and cells is vital to successful tissue engineering. In this study, we investigated the effects of a new fibrin sealant derived from snake venom as a scaffold for mesenchymal stem cells, to demonstrate the ability of cells to affect and detect the biological microenvironment. METHODS: The characterization of CD34, CD44 and CD90 expression on mesenchymal stem cells was performed by flow cytometry. In vitro growth and cell viability were evaluated by light and electron microscopy. Differentiation into osteogenic, adipogenic and chondrogenic lineages was induced. RESULTS: The fibrin sealant did not affect cell adhesion, proliferation or differentiation and allowed the adherence and growth of mesenchymal stem cells on its surface. Hoechst 33342 and propidium iodide staining demonstrated the viability of mesenchymal stem cells in contact with the fibrin sealant and the ability of the biomaterial to maintain cell survival. CONCLUSIONS: The new fibrin sealant is a three-dimensional scaffolding candidate that is capable of maintaining cell survival without interfering with differentiation, and might also be useful in drug delivery. Fibrin sealant has a low production cost, does not transmit infectious diseases from human blood and has properties of a suitable scaffold for stem cells because it permits the preparation of differentiated scaffolds that are suitable for every need.
Subject(s)
Fibrin Tissue Adhesive , Mesenchymal Stem Cells/cytology , Tissue Engineering/methods , Tissue Scaffolds/chemistry , Animals , Cell Adhesion/physiology , Cell Differentiation/physiology , Cell Proliferation/physiology , Flow Cytometry , Rats , Rats, Wistar , Snake VenomsABSTRACT
O objetivo principal da nossa pesquisa foi avaliar o potencial de diferenciação osteogênica de células-tronco mesenquimais (MSC) obtidas da medula óssea do cão. As MSC foram separadas pelo método Ficoll e cultivadas sob duas condições distintas: DMEM baixa glicose ou DMEM/F12, ambos contendo L-glutamina, 20% de SFB e antibióticos. Marcadores de MSC foram testados, confirmando células CD44+ e CD34- através da citometria de fluxo. Para a diferenciação osteogênica, as células foram submetidas a quatro diferentes condições: Grupo 1, as mesmas condições utilizadas para a cultura de células primárias com os meios DMEM baixa glicose suplementado; Grupo 2, as mesmas condições do Grupo 1, mais os indutores de diferenciação dexametasona, ácido ascórbico e b-glicerolfosfato; Grupo 3, células cultivadas com meios DMEM/F12 suplementado; e Grupo 4, nas mesmas condições que no Grupo 3, mais indutores de diferenciação de dexametasona, ácido ascórbico e b-glicerolfosfato. A diferenciação celular foi confirmada através da coloração com alizarin red e da imunomarcação com o anticorpo SP7/Osterix. Nós observamos através da coloração com alizarin red que o depósito de cálcio foi mais evidente nas células cultivadas em DMEM/F12. Além disso, usando a imunomarcação com o anticorpo SP/7Osterix obtivemos positividade em 1:6 células para o Meio DMEM/F12 comparada com 1:12 para o meio DMEM-baixa glicose. Com base nos nossos resultados concluímos que o meio DMEM/F12 é mais eficiente para a indução da diferenciação de células-tronco mesenquimais caninas em promotores osteogênicos. Este efeito provavelmente ocorre em decorrência da maior quantidade de glicose neste meio, bem como da presença de diversos aminoácidos.
The aim of our research was to evaluate the potential for osteogenic differentiation of mesenchimal stem cells (MSC) obtained from dog bone marrow. The MSC were separated using the Ficoll method and cultured under two different conditions: DMEM low glucose or DMEM/F12, both containing L-glutamine, 20% of FBS and antibiotics. MSC markers were tested, confirming CD44+ and CD34- cells with flow cytometry. For osteogenic differentiation, cells were submitted to four different conditions: Group 1, same conditions used for primary cell culture with DMEM supplemented media; Group 2, same conditions of Group 1 plus differentiation inductors Dexametazone, ascorbic acid and β-glicerolphosphate. Group 3, Cells cultured with supplemented DMEM/F12 media, and Group 4, same conditions as in Group 3 plus differentiation inductors Dexametazone, ascorbic acid and β-glicerolphosphate. The cellular differentiation was confirmed using alizarin red and imunostaining with SP7/Osterix antibody. We observed by alizarin staining that calcium deposit was more evident in cells cultivated in DMEM/F12.Furthermore, by SP/7Osterix antibody immunostaining we obtained 1:6 positive cells when using DMEM/F12 compared with 1:12 for low-glucose DMEM. Based on our results, we conclude that the medium DMEM/F12 is more efficient for induction of differentiation of mesenchymal stem cells in canine osteogenic progenitors. This effect is probably due to the greater amount of glucose in the medium and the presence of various amino acids.