APLICATII

In Vivo Angiogenesis Effect of Porous Collagen Scaffold with Hyaluronic Acid Oligosaccharides,
Cherng-Kang Perng, M.D.,*, Yng-Jiin Wang, Ph.D., Chi-Han Tsi, M.Sc.,* and Hsu Ma, M.D., Ph.D.*, Journal of Surgical Research -, 17 (2010)
INTRODUCTION

When injured, the human body deals with wounds through two types of reaction and repair processes: regeneration and healing [1]. With regeneration, the same cells and structures are produced to replace injured or missing tissue. A limited number of tissue typesepidermis and mucosa, for examplecan be regenerated in the human body. Healing, in which fibrous tissue replaces the original structures is more common in the human body. Here, the scar tissue generated by fibroblasts is functionally and structurally different from the original tissue. The most common example of healing by scarring is wound healing in human skin. Because the destroyed dermis cannot be regenerated, it is replaced by fibrous tissue. Angiogenesis is one of the most important steps in the process of tissue repair and wound healing. Granulation tissue, composed mainly of newly-formed vessels, can be found in wounds 4 d after injury [2]. Upon stimulation of growth factors produced by macrophages, endothelial cells from vessels in the adjacent tissue are activated, and then proliferate and migrate into wounds to become granulation tissue, which can bring in oxygen and nutrition for further healing process. When faced with a major injury and large tissue loss, the healing ability of the human body is usually not enough. Autologous or allogenic tissue or organ transplantation is presently the treatment of choice, but shortage of tissue sources is always a problem. Tissue engineering, which combines material science and cell

biology, is a promising solution for tissue defect repair. After decades of research and development, progress has been made, and some tissue engineering products, artificial skin, for example, has been commercialized and used clinically with some success. This success, however, has been limited to tissues with relatively simple structures that do not rely on sophisticated vascular systems to survive initially. Few breakthroughs have been made with complex 3-D tissue or organ engineering. One of the key problems here is how to grow and maintain complex tissue in vitro and keep the engineered tissue alive after implantation. Simple diffusion of oxygen and nutrition is not enough for these complex tissues, and adequate vascular systems must be built. Several methods are currently under investigation for promoting angiogenesis of tissue engineering constructs. These include scaffold design, the inclusion of angiogenic factors, in vivo prevascularization, and in vitro prevascularization [3]. It is well established that vascularization of implanted tissue-engineered constructs can be enhanced by adding angiogenic factors [4]. Because many of these growth factors are inherently unstable, systemic injection is not an effective strategy, as large amounts will be needed, leading to uncontrolled blood vessel formation at distant sites in the body [5]. A better approach is the incorporation of the growth factors into the tissue constructs, which guarantees localized delivery. In vivo prevascularization of engineered-tissue constructs is also known as tissue prefabrication. Here, the tissue construct is first implanted into a region with a vascular pedicle that can be used in the future microvascular transfer. A new vascular network will form from the vascular pedicle and vascularize the tissue construct [6]. Then, the tissue construct and the vascular system can be transferred together for reconstruction using a microsurgical technique. The downside to in vivo prevascularization is that the process is slow and the tissue construct may not fully vascularize. Another promising strategy is in vitro prevascularization. Here, a network of newly developed microvessels may be engineered in vitro by seeding scaffolds with endothelial cells [7]. After implantation, the ingrowing vessels can connect to the network and achieve faster revascularization of the tissue constructs. Scaffold design is also crucial to angiogenesis. Tissue engineering scaffolds, natural or synthetic, provide a 3-D framework for cells to attach to and grow in vitro, and also serve as a vehicle for transplantation. The ideal scaffold would be biocompatible and biodegradable and, more importantly, would exhibit good interaction with endothelial cells to promote angiogenesis. The structure of the biomaterial itself can affect angiogenesis. Fibroblasts cultured in a 3-D scaffold express more VEGF mRNA than monolayer culture [8]. The pore size of the scaffold also relates to angiogenesis; a diameter of 250 to 300 um is most effective for new vessel ingrowth [9]. Natural extracellular matrix proteins, such as collagen and glycosaminoglycan, have been used to construct

tissue engineering scaffolds. These scaffolds have proven to be more biocompatible than synthetic polymer scaffolds. Hyaluronic acid (HA), nonsulfated glycosaminoglycan, is a linear polysaccharide composed of a repeating disaccharide unit of (b, 1-4)-Dglucuronic acid and (b, 1-3)-N-acetyl-D-glucosamine, and is in its native state as a high-molecular weight polymer. Studies have shown that HA has a crucial effect in angiogenesis: high-molecular weight HA in its native form inhibits angiogenesis [10], but short chain HA with 3-10 disaccharide units are proangiogenic [11, 12]. Porous scaffolds of cross-linked type I collagen and high-molecular weight glycosaminoglycan have been used successfully for dermis tissue engineering [13, 14]. However, because the revascularization process is slower, these scaffolds are more prone to infection than conventional autologous skin transplantation. In this study, we synthesized porous scaffolds with cross-linked type I collagen and short-chain HA, and evaluated the angiogenesis effect of the scaffold in animal models. The purpose of the study is to compare the angiogenic properties such as the rate of vascularization between short chain and long chain HA when added to a porous scaffold with cross-linked type I collagen For years, cross-linked collagen-glycosaminoglycan porous scaffolds have been used as artificial skin for patients with extensive skin loss [16]. The scaffolds are designed to be biodegradable, as neodermis is formed underneath. Commercial versions of cross-linked collagenglycosaminoglycan porous scaffolds, such as Integra, have been shown to have high bacterial growth and wound infection when used in burn patients [17]. Wounds treated with Integra (Integra LifeSciences Corporation of Plainsboro, NJ) still suffered a certain percentage of deep or superficial infection [18] requiring topical or systemic treatment with antibiotics. Poor take and loss of Integra due to infection remains a concern for some patients [19]. With respect to conventional autologous skin graft, cross-linked collagen-glycosaminoglycan porous scaffolds require more time, up to 28 d [20], for full revascularization. High-molecular weight glycosaminoglycan in commercially available crosslinked collagen-glycosaminoglycan porous scaffolds is angiostatic. We used angiogenic low-molecular weight glycosaminoglycan, HA (MW 6.5 K), in cross-linked collagen-glycosaminoglycan porous scaffolds. As a result of improvements in the revascularization process of the scaffold achieved with short-chain HA, less wound infection and a higher graft take were expected. Angiogenesis in scaffolds with short-chain HA was observed as early as post-implantation day 14, and increased significantly at day 21and 28. Withlong-chainHA,angiogenesiswasnot observed until day 28.
Burke JF, Yannas IV, Quinby WC, Jr., et al. Successful use of a physiologically acceptable artificial skin in the treatment of extensive burn injury. Ann Surg 1981;194:413. 14. Heimbach D, Luterman A, Burke J, et al. Artificial dermis for major burns. A multi-center randomized clinical trial. Ann Surg 1988;208:313. 15. Bitter T, Muir HM. A modified uronic acid carbazole reaction. Anal Biochem 1962;4:330.

16. Yannas IV, Burke JF. Design of an artificial skin. I. Basic design principles. J Biomed Mater Res 1980;14:65. 17. Muangman P, Deubner H, Honari S, et al. Correlation of clinical outcome of Integra application with microbiologic and pathological biopsies. J Trauma 2006;61:1212. 18. Heimbach DM, Warden GD, Luterman A, et al. Multicenter postapproval clinical trial of Integra dermal regeneration template for burn treatment. J Burn Care Rehabil 2003;24:42. 19. Grant I, Green C, Martin R. Strategies to improve the take of commercially available collagen/glycosaminoglycan wound repair material investigated in an animal model. Burns 2001; 27:699. 20. Moiemen NS, Staiano JJ, Ojeh NO, et al. Reconstructive surgery with a dermal regeneration template: Clinical and histologic study. Plast Reconstr Surg 2001;108:93. 21. Slevin M, Krupinski J, Gaffney J, et al. Hyaluronan-mediated angiogenesis in vascular disease: Uncovering RHAMM

APLICATII Efectul in vivo Angiogeneza de poroase colagen schele cu Acid Hialuronic oligozaharide, Cherng-Kang Perng, MD, *, Wang Yng-Jiin, Ph.D., Tsi Chi-Han, Master, * i Ma Hsu, MD , Ph.D. *, Jurnalul de Cercetare chirurgicale -, 1-7 (2010) INTRODUCERE Atunci cnd accidentat, se ocup corpului uman cu rni prin intermediul a dou tipuri de procese de reacie i reparaii: regenerare i vindecare [1]. Cu regenerare, aceleai celule i structuri sunt produse pentru a nlocui vtmate sau lips esut. Un numr limitat de tesut tipurile de epiderm i-mucoasa, de exemplu-poate fi regenerat n corpul uman. Vindecare, n care fibroase esut nlocuiete structuri originale este mai frecvent n corpul uman. Aici, tesut cicatricial generate de fibroblastele este funcional i structural diferite din esutul original. Exemplul cel mai comun al vindecarea prin cicatrizare este vindecarea rnilor n pielea umana. Deoarece derma distruse nu pot fi regenerate, este nlocuit de esut fibros. Angiogeneza este unul dintre cei mai importani pai n Procesul de refacerea esuturilor i vindecarea rnilor. Granulare esut, compus n principal din navele nou-formate, pot fi gsite n rnile 4 d dup un prejudiciu [2]. La stimularea de factori de crestere produs de macrofage, celulelor endoteliale din vase n esutul adiacent sunt activate, i apoi prolifereaz i migreaz n rni s devin esutului de granulaie, care poate aduce n oxigen i nutriie pentru procesul de vindecare n continuare. Atunci cnd se confrunt cu un traumatism major i pierderi mari de esut, capacitatea de vindecare a corpului uman este de obicei, nu suficient. esuturi autologe sau allogenic sau transplantul de organe este n prezent un tratament de alegere, dar deficit de surse de tesut este ntotdeauna o problem. esut de inginerie, care combin tiina materialelor i de celule biologie, este o soluie promitoare pentru repararea tesuturilor defect. Dup decenii de cercetare i dezvoltare, progres a fost fcut, i unele produse de esut de inginerie, piele artificial, de exemplu, a fost comercializat punct de vedere clinic i utilizat cu un oarecare succes. Acest succes,

Cu toate acestea, a fost limitat la nivelul esuturilor cu relativ structuri simple care nu se bazeaz pe vasculare sofisticate sisteme pentru a supravieui iniial. Puine progrese au fost fcute cu esut complex de 3-D sau inginerie de organe. Una din problemele cheie aici este cum s creasc i s menin esuturi complexe in vitro i s pstreze proiectat n via dup implantare esut. Simplu de difuzie de oxigen i nutriie nu este suficient pentru aceste complexe esuturi, i sisteme adecvate vasculare trebuie s fie construite. Cteva metode sunt n prezent n curs de investigare pentru promovarea angiogeneza din ingineria tisular construiete. Acestea includ design schele, includerea de factori angiogenic, n prevascularization vivo, i n prevascularization vitro [3]. Este bine stabilit faptul c vascularizatie a implantat esut-inginerie construcii poate fi mbuntit prin adugarea de factori angiogenici [4]. Deoarece muli dintre aceti factori de cretere sunt n mod inerent injecie instabil, sistemic nu este un sistem eficient de strategie, ca sume mari vor fi necesare, care s conduc la necontrolat formarii vaselor de singe de la site-uri la distan n organism [5]. O abordare mai bun este ncorporarea factorii de cretere n esutul constructe, care garanteaz livrarea localizate. n prevascularization vivo de inginerie-esut constructe este, de asemenea cunoscut ca esut prefabricare. Aici, tesutul construi este, n primul implantat ntr-o regiune cu un pedicul vascular care poate fi utilizate n transferul microvasculare viitor. O nou reelei vasculare vor forma de la pediculului vascular i vasculariza tesutul construi [6]. Apoi, esut construi i sistemului vascular pot fi transferate mpreun pentru reconstructie folosind un microchirurgie tehnica. Dezavantajul la prevascularization vivo este c procesul este lent i esutul construi poate nu sunt pe deplin vasculariza. O alt strategie promitoare este n prevascularization vitro. Aici, o reea de nou microvessels dezvoltate pot fi proiectate in vitro de ctre Schele semnat cu celule endoteliale [7]. Dup implantare, navele ncarnat se poate conecta la reea i pentru a atinge revascularizare mai rapid a esut construiete. design schele este, de asemenea, crucial pentru angiogeneza. esut schele inginerie, natural sau sintetic, asigura un cadru 3-D pentru celule pentru a ataa i s creasc n vitro, i servesc, de asemenea, ca un vehicul pentru transplant. Ideal schel ar fi biocompatibile i biodegradabile i, mai important, ar expune bun interaciune cu celulelor endoteliale pentru a promova angiogeneza. Structura a biomaterialului n sine poate afecta angiogeneza. Fibroblastele cultivate ntr-un 3-D exprima schel mai VEGF ARNm dect cultur monostrat [8]. Dimensiunea porilor de

schel, de asemenea, se refer la angiogeneza, un diametru de 250 la 300 um este cel mai eficient pentru noi nave cresterea interna [9]. Naturale extracelulare proteine matriciale, cum ar fi colagenul i glicozaminoglican, au fost folosite pentru a construi Schele ingineria tisular. Aceste schele s-au dovedit a fi mai mult dect sintetice biocompatibile polimer constructii. Acidul hialuronic (HA), nonsulfated glicozaminoglican, este un polizaharid compus liniar de o unitate repetabil de dizaharide (b, 1-4)-Dglucuronic acid i (b, 1-3)-N-acetil-D-glucozamina, i este n stare nativa ca o mare greutate molecular polimer. Studiile au artat c HA are un rol crucial Efectul in angiogeneza: HA greutate molecular mare n forma sa original inhiba angiogeneza [10], dar de scurt lan HA cu 30 - 10 uniti de dizaharide sunt proangiogenic [11, 12]. Schele poroase de tip cross-linked I colagen i glicozaminoglican greutate molecular mare au fost folosite cu succes pentru ingineria tesuturilor dermul [13, 14]. Cu toate acestea, deoarece revascularizare Procesul este mai lent, aceste constructii sunt mult mai predispuse la infecie dect transplantul conventional pielea autolog. n acest studiu, am sintetizat schele poros cu tip cross-linked I colagen i cu lant scurt HA, i evaluat efectul angiogeneza a schelei pe modele animale. Scopul studiului este de a compara proprieti angiogenic, cum ar fi rata de vascularizatie ntre lan scurt i HA lan lung atunci cnd adugat la o schel poros de tip cross-linked I colagen De ani de zile, reticulate de colagen glicozaminoglican Schele poros au fost folosite ca pielea artificiala pentru pacienilor cu pierdere pielea extinse [16]. Schelelor sunt proiectate pentru a fi biodegradabile, ca neodermis se formeaz dedesubt. Versiunile comerciale de colagen reticulatglicozaminoglican schele poroase, cum ar fi Integra, s-au dovedit a avea o cretere ridicat bacteriene i infectarea plgilor atunci cnd este utilizat la pacienii cu arsuri [17]. Rnile tratai cu Integra (Integra LifeSciences Corporation a Plainsboro, NJ) a suferit nc o anumit Procentul de infecii profunde sau superficiale [18] care necesit tratamentul topic sau sistemic cu antibiotice. Srace s ia i pierderea de cauza infeciei Integra rmne o preocupare pentru unii pacieni [19]. n ceea ce privete convenionale Grefa de piele autolog, reticulate de colagen glicozaminoglican Schele poroase necesita mai mult timp, pn la 28 d [20], pentru revascularizare completa. High-greutate molecular glicozaminoglican n disponibile n comer reticulat schelele de colagen glicozaminoglican poros este angiostatic. Am folosit angiogenici greutate molecular mic glicozaminoglican, HA (MW 6.5 K), n cross-linked colagen-glicozaminoglican de constructii poroase. Ca un rezultat

de mbuntiri n procesul de revascularizare schel realizate cu lan scurt-HA, infectarea plgilor mai puin i o grefa mai mare ia fost de ateptat. Angiogeneza n schelele cu lan scurt-HA a fost observat nc din post-implantare 14-a zi, i a crescut n mod semnificativ la zi 21and 28. Withlong-chainHA, angiogenesiswasnot observate pn n ziua 28.