Biomimetic Delivery of Keratinocyte Growth Factor upon Cellular Demand for Accelerated Wound Healing in Vitro and in Vivo
2005; Elsevier BV; Volume: 167; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)61242-4
ISSN1525-2191
AutoresDavid J. Geer, Daniel D. Swartz, Stelios T. Andreadis,
Tópico(s)Tendon Structure and Treatment
ResumoExogenous 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. 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In addition to increased proliferation of basal cells, KGF induced proliferation in the normally quiescent suprabasal cell compartment and delayed differentiation.13Andreadis ST Hamoen KE Yarmush ML Morgan JR Keratinocyte growth factor induces hyperproliferation and delays differentiation in a skin equivalent model system.FASEB J. 2001; 15: 898-906Crossref PubMed Scopus (125) Google Scholar KGF is also important in protecting epithelial tissues from injury and apoptosis and promoting wound healing.14Finch PW Rubin JS Keratinocyte growth factor/fibroblast growth factor 7, a homeostatic factor with therapeutic potential for epithelial protection and repair.Adv Cancer Res. 2004; 91: 69-136Crossref PubMed Scopus (190) Google Scholar For example, KGF was strongly up-regulated in the intestines of patients suffering from bowel inflammatory disease15Brauchle M Madlener M Wagner AD Angermeyer K Lauer U Hofschneider PH Gregor M Werner S Keratinocyte growth factor is highly overexpressed in inflammatory bowel disease.Am J Pathol. 1996; 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Indeed, a recent study showed that a single injection of KGF DNA accelerated wound closure and reduced inflammation in a diabetic mouse model.37Marti G Ferguson M Wang J Byrnes C Dieb R Qaiser R Bonde P Duncan MD Harmon JW Electroporative transfection with KGF-1 DNA improves wound healing in a diabetic mouse model.Gene Ther. 2004; 11: 1780-1785Crossref PubMed Scopus (89) Google Scholar In general it has been difficult to maintain full bioactivity of the proteins applied in the wound space due to protein instability in the protease-rich environment of the wound.38Levy MY Barron LG Meyer KB Szoka Jr, FC Characterization of plasmid DNA transfer into mouse skeletal muscle: evaluation of uptake mechanism, expression and secretion of gene products into blood.Gene Ther. 1996; 3: 201-211PubMed Google Scholar, 39Choate KA Khavari PA Direct cutaneous gene delivery in a human genetic skin disease.Hum Gene Ther. 1997; 8: 1659-1665Crossref PubMed Scopus (78) Google Scholar In addition, bolus administration does not keep the protein localized and necessitates large amounts of growth factors that may have dangerous side effects, such as vascularization of nontarget tissues or growth of tumors.40Epstein SE Fuchs S Zhou YF Baffour R Kornowski R Therapeutic interventions for enhancing collateral development by administration of growth factors: basic principles, early results and potential hazards.Cardiovasc Res. 2001; 49: 532-542Crossref PubMed Scopus (178) Google Scholar The short biological half-life, lack of tissue-selectivity, and potential risk for carcinogenesis demand temporal and spatial control of growth factor delivery.41Putney SD Burke PA Improving protein therapeutics with sustained-release formulations.Nature Biotechnol. 1998; 16: 153-157Crossref PubMed Scopus (424) Google Scholar, 42Luginbuehl V Meinel L Merkle HP Gander B Localized delivery of growth factors for bone repair.Eur J Pharm Biopharm. 2004; 58: 197-208Crossref PubMed Scopus (272) Google Scholar To this end, biomaterials can be used to achieve controlled and localized release and at the same time serve as scaffolds to promote tissue regeneration. 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This approach was later extended to engineer fusion proteins containing the factor XIIIa recognition domain at the N-terminus and was used successfully to deliver β-nerve growth factor and vascular endothelial growth factor, in a manner that was controlled by cellular activity.46Zisch AH Schenk U Schense JC Sakiyama-Elbert SE Hubbell JA Covalently conjugated VEGF—fibrin matrices for endothelialization.J Control Release. 2001; 72: 101-113Crossref PubMed Scopus (306) Google Scholar, 50Sakiyama-Elbert SE Panitch A Hubbell JA Development of growth factor fusion proteins for cell-triggered drug delivery.FASEB J. 2001; 15: 1300-1302Crossref PubMed Scopus (148) Google Scholar We adopted this methodology to conjugate KGF into a fibrin matrix to achieve localized delivery that is in tune with cellular demand at the wound microenvironment. Instead of engineering a fusion protein, we covalently attached a peptide containing the α2-plasmin inhibitor fibrin-binding site to the free amines on the surface of the KGF molecule. The peptide-KGF complex (P-KGF) was conjugated to fibrin during polymerization and the binding efficiency depended strongly on the concentration of factor XIII. Plasmin digestion of the fibrin gels yielded KGF that was devoid of large fibrinogen fragments, detectable by gel electrophoresis. The residual grafted linker and small peptide fragment were not sufficient to alter KGF activity, as determined by promotion of epithelial cell proliferation. Using a scratch wound assay that we developed previously,51Geer DJ Andreadis ST A novel role of fibrin in epidermal healing: plasminogen-mediated migration and selective detachment of differentiated keratinocytes.J Invest Dermatol. 2003; 121: 1210-1216Crossref PubMed Scopus (59) Google Scholar we found that release of KGF via cell-mediated degradation of fibrin increased the rate of wound closure significantly and that KGF-induced healing was delayed by inhibition of fibrinolysis. To evaluate this delivery system in vivo we used a model system that we developed in our laboratory.52Geer DJ Swartz DD Andreadis ST Fibrin promotes migration in a three-dimensional in vitro model of wound regeneration.Tissue Eng. 2002; 8: 787-798Crossref PubMed Scopus (94) Google Scholar, 53Geer DJ Swartz DD Andreadis ST In vivo model of wound healing based on transplanted tissue-engineered skin.Tissue Eng. 2004; 10: 1006-1017Crossref PubMed Scopus (67) Google Scholar This system was based on transplantation of human skin equivalents onto full-thickness wound defects on the back of nude mice. In agreement with previous studies,54Medalie DA Tompkins RG Morgan JR Characterization of a composite tissue model that supports clonal growth of human melanocytes in vitro and in vivo.J Invest Dermatol. 1998; 111: 810-816Crossref PubMed Scopus (11) Google Scholar the transplanted tissues integrated well with the mouse skin and were infiltrated by dermal elements including fibroblasts and blood vessels to create a hybrid tissue comprising human epidermis and mouse dermis. At 6 weeks after transplantation, the humanized skin was subjected to a full thickness wound that was immediately treated with fibrin-bound KGF (Fb-P-KGF). Fluorescence imaging showed that Fb-P-KGF remained in the wound space for at least 7 days and enhanced re-epithelialization significantly. In agreement with our in vitro data, aprotinin delayed healing possibly by preventing fibrin degradation by the migrating cells. Taken together, our data shows that active KGF can be released from fibrin hydrogels in tune with cellular demand, providing localized treatment and enhancing tissue regeneration. Recombinant human KGF (6.25 mg/ml; Amgen, Thousand Oaks, CA) in 1× phosphate-buffered saline (PBS; Invitrogen, Carlsbad, CA) was reacted with succinimidyl trans-4-(maleimidylmethyl) cyclohexane-1-carboxylate (SMCC) (5 mg/ml; Molecular Probes, Eugene, OR) in dimethyl sulfoxide (Fisher Scientific, Pittsburgh, PA) at a 1:10 (SMCC:KGF) molar ratio for 1 to 1.5 hours (Figure 1A). Maleimido KGF was dialyzed against 500 ml of Tris-buffered saline (TBS; Invitrogen) at 4°C, using a 3500 molecular weight cutoff dialysis cassette (Slide-A-Lyzer; Pierce, Rockford, IL). TBS was changed every hour for a total of 3 hours. SMCC precipitate that formed during dialysis was removed by spinning the sample in centrifuge at 16,000 × g for 5 minutes. A peptide having the sequence, (Flc)-LNQEQVSPRKKC [(Flc) = fluorescein], was synthesized (Sigma Genosys, The Woodlands, TX) and contained the α2-plasmin inhibitor motif, NQEQVSP for binding to fibrin gels as described before.48Schense JC Hubbell JA Cross-linking exogenous bifunctional peptides into fibrin gels with factor XIIIa.Bioconjug Chem. 1999; 10: 75-81Crossref PubMed Scopus (261) Google Scholar The peptide (2.5 mg/ml in PBS) was then reacted with the derivatized KGF at the indicated molar ratio for 1 hour at room temperature then overnight at 4°C. Using a 10,000 molecular weight cutoff dialysis cassette (Pierce), KGF with bound peptide (P-KGF) was purified from unbound peptide by dialysis against 500 ml of TBS at 4°C. TBS was changed every hour for a total of 3 hours. Final concentration of P-KGF was determined enzymatically using an enzyme-linked immunosorbent assay (ELISA) as discussed below. Different KGF unknowns were diluted with sample buffer [0.0625 mol/L Tris-HCl, 25% (v/v) glycerol, 2% (w/v) sodium dodecyl sulfate, 0.01% bromophenol blue, and 5% (v/v) β-mercaptoethanol] and heated at 95°C for 5 minutes. The KGF samples were then placed on ice for 10 minutes before they were loaded onto a 14% denaturing gel (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and run for 45 minutes. The proteins were then transferred onto a polyvinylidene difluoride membrane (Immun-Blot PVDF; Bio-Rad Laboratories, Hercules, CA) for 1 hour using an electrophoretic transfer cell (Mini Trans-Blot, Bio-Rad Laboratories, Hercules, CA). The membrane was incubated in blocking agent [5% (w/v) nonfat milk in wash buffer (TBS/0.1% Tween 20)] overnight at 4°C. The next day, the membrane was incubated with goat polyclonal anti-KGF (R&D Systems, Minneapolis, MN) at a concentration of 0.1 μg/ml in blocking agent. The membrane was washed five times with wash buffer and incubated with horseradish peroxidase-conjugated mouse anti-goat IgG polyclonal secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) at 0.2 μg/ml in blocking agent for 1 hour at room temperature. The membrane was washed five times with wash buffer, and the bands were detected using chemiluminescence (LumiGLO; KPL, Gaithersburg, MD) according to the manufacturer's instructions. Fibrin gels were prepared by mixing two solutions: one containing fibrinogen (6.25 mg/ml; Sigma, St. Louis, MO) or highly purified fibrinogen (6.25 mg/ml; Enzyme Research Laboratories, South Bend, IN), P-KGF (100 to 500 μg/ml) and factor XIII (1.25 to 12.5 PEU/ml, Enzyme Research Laboratories) and the other containing thrombin (12.5 U/ml, Sigma) and calcium chloride (12.5 mmol/L, Sigma) in 1× TBS. The two solutions were mixed in a 4:1 volumetric ratio to make a fibrin gel containing 5 mg/ml fibrinogen, 2.5 U/ml thrombin, 2.5 mmol/L calcium chloride, 1 to 10 PEU/ml factor XIII, and 100 to 500 μg/ml of P-KGF. For some experiments, the plasmin inhibitor aprotinin (0 to 1000 kIU/ml, Sigma) was added to the thrombin-containing fraction at the indicated concentration. To assess binding efficiency, gels were allowed to fully polymerize at 37°C for 30 minutes then submerged in wash buffer (TBS) at 4°C. The wash buffer was collected at indicated times after polymerization for measuring the release of unbound growth factor as a function of time and then replaced with fresh buffer. After 15 hours the gels were degraded by incubation with plasmin (0.25 U/ml in TBS; Calbiochem, San Diego, CA) for 3 hours at 37°C. An ELISA was used to quantify the amount of KGF recovered from the fibrin gels. The wells of 96-well ELISA plates were coated (100 μl/well) with 1.0 μg/ml of monoclonal mouse anti-human KGF antibody (R&D Systems) in PBS overnight at 4°C. The next day, the plates were washed three times with PBS/0.5% Tween-20 (Sigma) and nonspecific binding sites were blocked by incubation with PBS/10% horse serum (Invitrogen) for 1 hour at room temperature. A standard curve was generated with stock KGF (0.5 to 16 ng/ml) as recommended by the manufacturer (R&D Systems). Unknown KGF samples were serially diluted in PBS/1% bovine serum albumin and incubated (100 μl/well) for 1 hour at room temperature. The plates were then washed three times with PBS/0.5% Tween-20 and polyclonal goat anti-human KGF (R&D Systems) was added (100 μl/well) at a concentration of 1.0 μg/ml in PBS/10% horse serum for 1 hour at room temperature. After three washes with PBS/0.5% Tween-20, horseradish peroxidase-conjugated mouse anti-goat IgG secondary antibody (Jackson ImmunoResearch Laboratories) was incubated (100 μl/well) at a concentration of 1.0 μg/ml for 1 hour at room temperature. Plates were washed three times with PBS/0.5% Tween-20 and substrate was added (100 μl/well) (Sigma Fast o-phenylenediaminedihydrochloride tablet sets; Sigma). The reaction was allowed to proceed for 3 to 5 minutes before the addition of 50 μl per well of 4 mol/L H2SO4. The OD490 was measured with an absorbance microplate reader (SpectraMax 340; Molecular Devices, Menlo Park, CA). To assess total recovered fluorescence, 100 μl of each sample was removed and placed in a black 96-well plate. The fluorescence intensity (excitation, 490 nm; emission, 520 nm) was measured in a fluorescence microplate reader (SpectraMax Gemini, Molecular Devices). A standard curve was generated by serially diluting stock P-KGF. The intensity of degraded fibrin without P-KGF was subtracted as background. A growth assay was used to assess functionality of KGF after chemical modifications. Rhesus
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