Biomimetic Delivery of Keratinocyte Growth Factor upon Cellular Demand for Accelerated Wound Healing in Vitro and in Vivo

Abstract

Exogenous keratinocyte growth factor (KGF) significantly enhances wound healing, but its use is hampered by a short biological half-life and lack of tissue selectivity. We used a biomimetic approach to achieve cell-controlled delivery of KGF by covalently attaching a fluorescent matrix-binding peptide that contained two domains: one recognized by factor XIII and the other by plasmin. Modified KGF was incorporated into the fibrin matrix at high concentration in a factor XIII-dependent manner. Cell-mediated activation of plasminogen to plasmin degraded the fibrin matrix and cleaved the peptides, releasing active KGF to the local microenvironment and enhancing epithelial cell proliferation and migration. To demonstrate in vivo effectiveness, we used a hybrid model of wound healing that involved transplanting human bioengineered skin onto athymic mice. At 6 weeks after grafting, the transplanted tissues underwent full thickness wounding and treatment with fibrin gels containing bound KGF. In contrast to topical KGF, fibrin-bound KGF persisted in the wounds for several days and was released gradually, resulting in significantly enhanced wound closure. A fibrinolytic inhibitor prevented this healing, indicating the requirement for cell-mediated fibrin degradation to release KGF. In conclusion, this biomimetic approach of localized, cell-controlled delivery of growth factors may accelerate healing of large full-thickness wounds and chronic wounds that are notoriously difficult to heal. Exogenous keratinocyte growth factor (KGF) significantly enhances wound healing, but its use is hampered by a short biological half-life and lack of tissue selectivity. We used a biomimetic approach to achieve cell-controlled delivery of KGF by covalently attaching a fluorescent matrix-binding peptide that contained two domains: one recognized by factor XIII and the other by plasmin. Modified KGF was incorporated into the fibrin matrix at high concentration in a factor XIII-dependent manner. Cell-mediated activation of plasminogen to plasmin degraded the fibrin matrix and cleaved the peptides, releasing active KGF to the local microenvironment and enhancing epithelial cell proliferation and migration. To demonstrate in vivo effectiveness, we used a hybrid model of wound healing that involved transplanting human bioengineered skin onto athymic mice. At 6 weeks after grafting, the transplanted tissues underwent full thickness wounding and treatment with fibrin gels containing bound KGF. In contrast to topical KGF, fibrin-bound KGF persisted in the wounds for several days and was released gradually, resulting in significantly enhanced wound closure. A fibrinolytic inhibitor prevented this healing, indicating the requirement for cell-mediated fibrin degradation to release KGF. In conclusion, this biomimetic approach of localized, cell-controlled delivery of growth factors may accelerate healing of large full-thickness wounds and chronic wounds that are notoriously difficult to heal. Re-establishing an epithelial barrier in injured skin is a crucial wound-healing event that protects the body against further water loss and exposure to external pathogens. Skin epithelial cells or keratinocytes begin migrating to repair the epithelium ∼18 to 24 hours after injury1Coulombe PA Towards a molecular definition of keratinocyte activation after acute injury to stratified epithelia.Biochem Biophys Res Commun. 1997; 236: 231-238Crossref PubMed Scopus (111) Google Scholar carefully degrading a fibrin-rich clot through the activation of plasminogen into plasmin.2Collen D Ham-Wasserman lecture: role of the plasminogen system in fibrin-homeostasis and tissue remodeling.Hematology (Am Soc Hematol Educ Program). 2001; : 1-9Crossref PubMed Scopus (79) Google Scholar Transient interactions with extracellular matrix proteins in the wound such as collagen and fibronectin during fibrinolysis of the clot guide the keratinocytes into the wound space. Degranulating platelets are embedded in the fibrin matrix and release growth factors and cytokines, which activate keratinocytes to proliferate and migrate until they heal the defect. Keratinocyte growth factor (KGF), a member of the fibroblast growth factor family (FGF-7), plays a prominent role in epithelial morphogenesis and wound healing.3Aaronson SA Bottaro DP Miki T Ron D Finch PW Fleming TP Ahn J Taylor WG Rubin JS Keratinocyte growth factor. A fibroblast growth factor family member with unusual target cell specificity.Ann NY Acad Sci. 1991; 638: 62-77Crossref PubMed Scopus (136) Google Scholar, 4Werner S Keratinocyte growth factor: a unique player in epithelial repair processes.Cytokine Growth Factor Rev. 1998; 9: 153-165Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar Although initial studies restricted expression of KGF in cells of mesenchymal origin,5Rubin JS Bottaro DP Chedid M Miki T Ron D Cheon G Taylor WG Fortney E Sakata H Finch PW LaRochelle WJ Keratinocyte growth factor.Cell Biol Int. 1995; 19: 399-411Crossref PubMed Scopus (258) Google Scholar, 6Rubin JS Bottaro DP Chedid M Miki T Ron D Cunha GR Finch PW Keratinocyte growth factor as a cytokine that mediates mesenchymal-epithelial interaction.EXS. 1995; 74: 191-214PubMed Google Scholar a recent study documented expression of KGF by dendritic epidermal T cells of the skin immediately after wounding,7Jameson J Ugarte K Chen N Yachi P Fuchs E Boismenu R Havran WL A role for skin gammadelta T cells in wound repair.Science. 2002; 296: 747-749Crossref PubMed Scopus (500) Google Scholar suggesting that KGF may play an important role in epidermal immunity. The paracrine action of KGF on epithelial cells is mediated through the KGF receptor (KGFR or FGFR2IIIb), a splice variant of FGF-2 receptor encoded by the gene fgfr-2.8Miki T Fleming TP Bottaro DP Rubin JS Ron D Aaronson SA Expression cDNA cloning of the KGF receptor by creation of a transforming autocrine loop.Science. 1991; 251: 72-75Crossref PubMed Scopus (360) Google Scholar, 9Miki T Bottaro DP Fleming TP Smith CL Burgess WH Chan AM Aaronson SA Determination of ligand-binding specificity by alternative splicing: two distinct growth factor receptors encoded by a single Acad PubMed Scopus Google Scholar KGF is to an important player in and morphogenesis of epithelial tissues and a of mesenchymal-epithelial PW Cunha GR Rubin JS J Ron D of keratinocyte growth factor and keratinocyte growth factor receptor expression during a role in mesenchymal-epithelial 1995; PubMed Scopus Google Scholar, Smith R growth expression during in and PubMed Scopus Google Scholar KGF expression to the keratinocytes of epidermal and the of epidermal Fuchs E expression of keratinocyte growth factor to keratinocytes in epithelial in PubMed Scopus Google Scholar of with keratinocytes in and of the of the In to proliferation of KGF proliferation in the cell and Keratinocyte growth factor and in a skin model 2001; PubMed Scopus Google Scholar KGF is important in epithelial tissues injury and and wound PW Rubin JS Keratinocyte growth growth factor a factor with for epithelial and PubMed Scopus Google Scholar KGF was in the of M M K M S Keratinocyte growth factor is in J Google Scholar, PW A expression of keratinocyte growth factor with Full Text PDF PubMed Scopus Google Scholar and the epithelium K Fuchs E Havran WL Boismenu R of the by T Acad 2002; PubMed Scopus Google Scholar several studies that KGF the epithelium Rubin JS Smith of keratinocyte growth factor in 1995; PubMed Scopus Google Scholar, M H KGF repair of in epithelial J 1997; Google Scholar, J AM S P keratinocyte growth factor protects against J 1998; Google Scholar, P M Chen A expression of keratinocyte growth factor (KGF) in epithelial cell by Acad PubMed Scopus Google Scholar by expression of K Rubin JS Keratinocyte growth factor for and in cells in J 1995; Google Scholar of M AM Keratinocyte growth factor in J 1997; PubMed Scopus Google Scholar and N Keratinocyte growth factor epithelial cell repair after J 1998; Google Scholar KGF was to and and injury and CL Chen S AM A Keratinocyte growth factor protects and injury and 1998; Google Scholar, CL SA Chen S of keratinocyte growth factor in the epithelium of the of and J PubMed Scopus Google Scholar, CL Chen SA A S The of keratinocyte growth factor in of 2002; PubMed Scopus Google Scholar a are in to the of KGF on in the a that or treatment PW Rubin JS Keratinocyte growth growth factor a factor with for epithelial and PubMed Scopus Google Scholar In KGF is after suggesting that KGF may crucial in of the healing S of keratinocyte growth factor expression in the during wound Acad PubMed Scopus Google Scholar KGF at and proliferation of keratinocytes at the wound was Fuchs E Keratinocyte growth factor is for but for wound PubMed Scopus Google Scholar to the action of other of the J S fibroblast growth factor cDNA and of 1997; PubMed Scopus Google Scholar or S R growth factor and its role during skin and PubMed Scopus Google Scholar biological KGF significantly enhanced in full and thickness wounds in and wound Rubin JS S T A G M A Aaronson SA keratinocyte growth factor in a model of epidermal wound PubMed Scopus Google Scholar, D K E of epithelial during skin by keratinocyte growth PubMed Scopus Google Scholar In to KGF tissue in an wound through the of Keratinocyte growth factor tissue in of cell PubMed Scopus Google Scholar expression of KGF was in S M G of keratinocyte growth factor expression is and during wound healing in the PubMed Scopus Google Scholar, M R S of keratinocyte growth factor expression by in and during wound 1995; PubMed Scopus Google Scholar suggesting that KGF may of chronic wounds that are notoriously difficult to heal. a recent study that a single of KGF wound and in a G M J R R P with wound healing in a PubMed Scopus Google Scholar In difficult to full of the proteins in the wound to in the of the of into of expression and of gene into Google Scholar, PA gene delivery in a human skin 1997; PubMed Scopus Google Scholar In the and large of growth factors that may such as of tissues or growth of Fuchs S R R for enhancing by of growth and 2001; PubMed Scopus Google Scholar The short biological lack of and for and of growth factor PA with 1998; PubMed Scopus Google Scholar, delivery of growth factors for J PubMed Scopus Google Scholar To this used to achieve and release and at the as to tissue studies used to growth factors or for of tissues N T H N J M H K K A as a cytokine delivery system for 2001; 19: Scopus Google Scholar release of growth factor a cell PubMed Scopus Google Scholar as a gene delivery enhances wound PubMed Scopus Google Scholar and for 2001; PubMed Scopus Google Scholar, M N H D P release of cell in growth for tissue Google Scholar In was that used to covalently cell into fibrin gels with factor PubMed Scopus Google Scholar, of is mediated by and Biol PubMed Scopus Google Scholar or growth such as growth factor or into fibrin release of growth factor a cell PubMed Scopus Google Scholar the one of the peptide was recognized by factor and incorporated into fibrin during the which in with the growth factor. approach was to proteins containing the factor at the and was used to growth factor and growth in a that was by for 2001; PubMed Scopus Google Scholar, A of growth factor proteins for 2001; PubMed Scopus Google Scholar We this to KGF into a fibrin matrix to achieve delivery that is in with at the wound of a we covalently a peptide containing the inhibitor to the on the of the KGF The was to fibrin during and the on the concentration of factor of the fibrin gels KGF that was of large by The and peptide to KGF as by of epithelial cell a wound that we A role of fibrin in epidermal and of PubMed Scopus Google Scholar we that release of KGF cell-mediated degradation of fibrin the of wound significantly and that healing was by of To this delivery system in vivo we used a model system that we in in a in model of wound 2002; PubMed Scopus Google Scholar, In vivo model of wound healing on transplanted PubMed Scopus Google Scholar system was on of human skin onto full-thickness wound on the of mice. In with of a tissue model that growth of human in and in 1998; PubMed Scopus Google Scholar the transplanted tissues with the skin and by and to a hybrid tissue human and At 6 weeks after the skin was to a full thickness wound that was immediately with fibrin-bound KGF that in the wound for at days and enhanced In with in healing by fibrin degradation by the migrating that active KGF released fibrin in with treatment and enhancing tissue human KGF in was with in at a for to hours KGF was against of at a molecular was for a of that during was by the in at for A peptide the was The and contained the inhibitor for to fibrin gels as into fibrin gels with factor PubMed Scopus Google Scholar The peptide in was with the KGF at the for at at a molecular KGF with bound peptide was peptide by against of at was for a of concentration of was an as KGF with and and at for The KGF on for they onto a and for The proteins onto a for an cell The was in in at The the was with at a concentration of in The was with and with at in for at The was with and the to the gels by two one containing or to and factor XIII to and the other containing and in The two in a to a fibrin containing to factor and to of the plasmin inhibitor to was to the at the To gels to at for in at The was at after for the release of growth factor as a of and with hours the gels degraded by with plasmin in for hours at was used to the of KGF the fibrin The of with of KGF in at The the with and by with for at A was with KGF to as by the KGF in and for at The with and KGF was at a concentration of in for at with was at a concentration of for at with and was The was to for to the of of The was with an To of was and in a The was in a A was by The of degraded fibrin was as A growth was used to of KGF after epithelial cells they to to KGF and epidermal growth JS H Finch PW Taylor WG S Aaronson SA and of a growth factor for epithelial Acad PubMed Scopus Google Scholar The cells in with epidermal growth factor and was to days and cells they the growth with of collagen for hours at of the KGF in that of with and and in an and as and and in at a concentration of onto the KGF and in the The of cells at was a as A role of fibrin in epidermal and of PubMed Scopus Google Scholar cells in and to in with and of epidermal growth factor. days the with a a wound in after with to cell and fibrin gels or the wound and gels containing of plasminogen and of in to after with was to the of keratinocytes and on of as in a in model of wound 2002; PubMed Scopus Google Scholar used for of skin one to and of skin as A role of fibrin in epidermal and of PubMed Scopus Google Scholar At weeks after and at 6 weeks the and in the with a The full thickness wound the was with as and to the wound they to for a large of was the wound and with At by an of as in the and in or for or of the wounds a of with a was and into with a tissue The wound with to fluorescent of the gels during healing of the skin a fluorescent we the wounds a in a that was to an and for and a was with and and of tissue A of of the wounds at after wounding at on an a and of of the wounds was by the of the to the of the initial as the in the as A role of fibrin in epidermal and of PubMed Scopus Google Scholar of the a We used fibrin gels to KGF on to wound healing in and in To this we KGF to the peptide which the for factor XIII and incorporated into fibrin gels during into fibrin gels with factor PubMed Scopus Google Scholar, of is mediated by and Biol PubMed Scopus Google Scholar, J P of into fibrin enhances Scopus Google Scholar The was to to of the bound KGF The peptide was to KGF through a that was at the of a In the KGF was with a a the was with the peptide a the of the peptide and the on the KGF The in loss of growth factor as by a KGF of the that the of KGF on the initial of peptide to KGF. at of the contained a of with to KGF at a the growth factor was with a of two KGF was with of and and with in the of of factor The gels and degraded with plasmin to release KGF as by the of fibrin clot degradation during wound that KGF degraded gels contained peptide and suggesting that plasmin may at the treatment of to fibrin with plasmin a with molecular as KGF To the of that was bound to the gels to release and degraded with plasmin. The was as the of initial we that the was on factor XIII and at a was used that was but contained such as fibronectin and the was at of factor at a high concentration of factor XIII the as with suggesting that may by the of factor the of the the of by to the high of the fibrin we the of KGF release the fibrin gels an In with the or of the growth factor was released the gels containing or of factor In gels containing KGF with released of the growth factor 24 In of the growth factor was released in and of the KGF was released 6 The biological of KGF was by proliferation of epithelial cells which are to to epidermal growth factor and JS H Finch PW Taylor WG S Aaronson SA and of a growth factor for epithelial Acad PubMed Scopus Google Scholar and proliferation of cells to a as KGF, suggesting that with peptides, to fibrin and release through the action of plasmin on biological of KGF In the of cells in to cell In with M M K M S Keratinocyte growth factor is in J Google Scholar, PW A expression of keratinocyte growth factor with Full Text PDF PubMed Scopus Google Scholar, K Fuchs E Havran WL Boismenu R of the by T Acad 2002; PubMed Scopus Google Scholar, Rubin JS Smith of keratinocyte growth factor in 1995; PubMed Scopus Google Scholar, M H KGF repair of in epithelial J 1997; Google Scholar, J AM S P keratinocyte growth factor protects against J 1998; Google Scholar, P M Chen A expression of keratinocyte growth factor (KGF) in epithelial cell by Acad PubMed Scopus Google Scholar that in to KGF, and may cell The of on wound healing was a wound that we A role of fibrin in epidermal and of PubMed Scopus Google Scholar In this a cell was collagen and fibrin to the that epithelial cells during wound healing in they migrate the collagen of the and the fibrin clot in the wound for tissue R R of Scholar To this cells on they the and with which to after to the cell The fibrin gels contained factor XIII and of as We that wound healing cell by after hours in the of as to with fibrin and at degradation of the fibrin gels and wound healing in a manner. In the of fibrin gels degraded to and healing was At of the fibrin gels degraded hours and the of healing of prevented of fibrin gels and wound demonstrate that degradation of fibrin by the migrating cells may to release KGF and accelerate wound To the of in we used an in vivo wound model that we in In vivo model of wound healing on transplanted PubMed Scopus Google Scholar model was on bioengineered human that was transplanted onto athymic mice. 6 weeks after the tissues with and and with the skin to a hybrid skin tissue with human and the to full-thickness wounds they with as human suggesting that this may a model of human epidermal In vivo model of wound healing on transplanted PubMed Scopus Google Scholar To the of in wound healing, transplanted skin at 6 weeks after grafting, and the wounds immediately with a single of KGF with fibrin gels or we was at the wound at and days after wounding A and At was in the indicating release of the growth factor the matrix into the wound In fibrin the wound at and of the tissues at days after wounding epidermal cells migrating on and degrading the fibrin with fluorescent of the tissues significantly healing of the wounds with and and as to of tissue and of tissue in the wounds with and and as to of Skin to athymic and to for 6 weeks to a full-thickness At days after tissues and for with or A of was to of the wound and of wound the of the migrating and are by and fibrin of the migrating that wounds with or by To fibrin degradation was for KGF we the concentration by to to in suggesting that was for fibrin degradation KGF was released by the In at high concentration of healing of wounds was to the of that release of KGF fibrin degradation by the that of fibrinolysis may the of wound healing in In this study we used a growth factor delivery system that local delivery of KGF on to wound To this we the growth factor to fibrin by to the of the In we the of KGF to the of release and the biological after plasmin of fibrin We further the of this system in vivo a model of wound healing on transplanted human bioengineered in the wound for several in the of the fibrin matrix for the growth factor at the wound cells into the wound they degraded the fibrin releasing KGF cell-mediated release in enhanced and tissue the release of bound but KGF, suggesting that degradation of may for enhanced healing of to KGF was a that of the of KGF KGF several that with to a for with the several incorporated to the of the the that at one peptide to factor KGF in its active peptide the biological of the as by the growth and and growth factors or with in active of may to that the peptide proteins containing a growth factor with the peptide at the fibrin gels in a cell-controlled for 2001; PubMed Scopus Google Scholar, M N H D P release of cell in growth for tissue Google Scholar, A of growth factor proteins for 2001; PubMed Scopus Google Scholar the of the may the is the or as is the with and S A target for with the growth proliferation and collagen Acad PubMed Scopus Google Scholar, H J A J of the growth factor of the growth factor receptor by Full Text Full Text PDF PubMed Scopus Google Scholar the of peptide on the of with and the of the of in of incorporated into fibrin in the of other proteins that may for into the S N in for Biol Full Text PDF PubMed Google Scholar A high concentration of factor XIII was to of fibrin which molecular or the of by the of factor with growth factor. a release of growth factor to a by release of the factor on fibrin degradation by the is to a peptide to or or D Ham-Wasserman lecture: role of the plasminogen system in fibrin-homeostasis and tissue remodeling.Hematology (Am Soc Hematol Educ Program). 2001; : 1-9Crossref PubMed Scopus (79) Google Scholar such as A of growth factor proteins for 2001; PubMed Scopus Google Scholar or M A P cell proliferation and by fibrin-bound with to local PubMed Scopus Google Scholar into the peptide and to for release In that treatment with plasmin the suggesting that the may to is important to that the to the the of KGF was proliferation and studies with epithelial cells that of the peptide and treatment with plasmin the biological of KGF. In KGF that was released by treatment with plasmin its biological suggesting that this may in this as healing of human skin that transplanted onto athymic mice. this model system several of the of human the of and the of In vivo model of wound healing on transplanted PubMed Scopus Google Scholar suggesting that the of may to human wound of degradation of fibrin gels and wound healing in and in suggesting that fibrin degradation may for release of and wound The of fibrin degradation the and of growth factor the of wound studies are to healing is at of The are to on the of wound chronic wounds P for skin 1997; PubMed Scopus Google Scholar, The of chronic PubMed Scopus Google Scholar, K P of single cells to wounds in PubMed Scopus Google Scholar and may of for fibrin At the concentration of KGF used in this study and in wound healing in is that may the of healing or may at KGF. KGF the of delivery with on a with for use in treatment of In contrast to of release that on molecular release of KGF fibrin is in with and is restricted to the local microenvironment of the migrating KGF in the wound until migrating cells the fibrin matrix as we by of the gels in action is to the for the of degradation and the of KGF, on D but in Full Text PDF PubMed Scopus Google Scholar, R gene delivery to of PubMed Scopus Google Scholar is important in of several studies that that or the target the of large of growth M E of fibroblast growth factor: of and 1997; PubMed Scopus Google Scholar, mediated gene in wound PubMed Scopus Google Scholar, Burgess of growth factor release a 2002; PubMed Scopus Google Scholar for the that the of or cells was significantly and or wounds and this by the of KGF A may that at days after wounding cell proliferation may in and wounds they In wounds by and keratinocytes and migrating to the KGF in the of the healing S of keratinocyte growth factor expression in the during wound Acad PubMed Scopus Google Scholar is that and and proliferation at A study to the of and on and epidermal The of in in vivo model that this may used for treatment of which are with fibrin and use of fibrin in 1995; PubMed Scopus Google Scholar, D of and on the of injured to human cell 1997; PubMed Scopus Google Scholar, K K A in fibrin into Scopus Google Scholar delivery to in the body with to and the two and in of delivery may for the treatment of chronic wounds such as in which KGF may but as through of growth P Keratinocyte growth factor and protects barrier Sci. Google Scholar expression of KGF is to in the wounds of S M G of keratinocyte growth factor expression is and during wound healing in the PubMed Scopus Google Scholar, M R S of keratinocyte growth factor expression by in and during wound 1995; PubMed Scopus Google Scholar cell-controlled delivery to in as to acute wounds are to high suggesting that used to the release of or other growth factors for with KGF the matrix on The that we used is and used to growth factor into the fibrin The large of fibrin gels of growth factors that of the healing growth factor and growth which recent for of chronic S K D K S K T growth factor wound healing in a PubMed Scopus Google Scholar, J Taylor of growth factor on wound in the PubMed Scopus Google Scholar, M D D N H M human growth factor gene and wound healing in the Google Scholar, S A G G G M gene enhances wound healing by in 2002; 9: PubMed Scopus Google Scholar the used for delivery of or to achieve gene into the wound space. The of cells to may gene the for such as G M J R R P with wound healing in a PubMed Scopus Google Scholar of bioengineered human skin onto a model system that used to in a in vivo We for the of KGF.