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. 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 ϳ18 to 24 hours after injury 1 carefully degrading a fibrin-rich clot through the activation of plasminogen into plasmin. 2 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. 3,4 Although initial studies restricted expression of KGF in cells of mesenchymal origin, 5,6 a recent study documented expression of KGF by dendritic epidermal T cells of the skin immediately after wounding, 7 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. 8,9 KGF is thought to be an important player in development and morphogenesis of epithelial tissues and a promoter of mesenchymal-epithelial interactions. 10,11 Targeting KGF expression to the basal keratinocyt...

Vascularization of the Dermal Support Enhances Wound Re-Epithelialization by In Situ Delivery of Epidermal Keratinocytes

“…When keratinocytes are seeded on the basement membrane side of the dermis and raised to the air-liquid interface, they differentiate to form a fully stratified epidermis with basal, suprabasal, granular, and cornified cell layers exhibiting barrier function. 10,11 In contrast to tissue constructs based on hydrogels such as collagen or fibrin, the dermis retains the mechanical strength and elasticity of human skin; therefore, it is easy to handle during transplantation. Thus, acellular dermis offers an effective off-the-shelf dressing for immediate coverage of wounds to avoid infection and reduce water loss.…”

“…3,11,28 To infiltrate the dermis with HaCaT-conditioned medium, a piece of 1.5 cm 2 acellular dermis was placed basement membrane side down in a vacuum device. Then, the dermis was infiltrated with a mixture of 200 mL of thrombin (2.5 mU/mL; Sigma) and 80 mL of HaCaT-conditioned medium for 1 min, followed by 200 mL of fibrinogen (8 mg/mL; Enzyme Research Laboratories) for 5 min or until a thin layer of fibrin was visible at the bottom of the dermis.…”

“…11,25,26,28 The presence of keratin-10, laminin, KGF, and proliferating cell nuclear antigen (PCNA) in the tissue samples was detected in TFM-embedded tissues by immunohistochemistry. Briefly, the cryosections were washed twice in phosphate-buffered saline (PBS) followed by blocking in 10% goat serum/PBS for 1 h at room temperature.…”

“…3,4 On the other hand, chronic wounds, which commonly occur in diabetic patients, also require special medical intervention often resulting in drastic surgical procedures such as amputation of the infected limb. Despite intense efforts devoted into development of tissue-engineered skin, [5][6][7][8][9][10][11][12][13] the autologous skin graft remains the gold standard for skin replacement.…”

Impact of combinatory growth factor application on rabbit Achilles tendon injury with operative versus conservative treatment: A pilot study

“…Follow-up studies of this pilot study should aim to overcome the short biological half-life and early degradation of the growth factors, e.g., by embedding them in slow release fibrin and gelatin matrices (30) or microbeads (31), which are degraded later and release biologically active growth factors after the inflammatory response has ended.…”