Spontaneous skin erosions and reduced skin and corneal wound healing characterize CLIC4(NULL) mice

Cutaneous wound healing is a complex process involving blood clotting, inflammation, migration of keratinocytes, angiogenesis, and, ultimately, tissue remodeling and wound closure. Many of these processes involve transforming growth factor-β (TGF-β) signaling, and mice lacking components of the TGF-β signaling pathway are defective in wound healing. We show herein that CLIC4, an integral component of the TGF-β pathway, is highly up-regulated in skin wounds. We genetically deleted murine CLIC4 and generated a colony on a C57Bl/6 background. CLIC4(NULL) mice were viable and fertile but had smaller litters than did wild-type mice. After 6 months of age, up to 40% of null mice developed spontaneous skin erosions. Reepithelialization of induced full-thickness skin wounds and superficial corneal wounds was delayed in CLIC4(NULL) mice, resolution of inflammation was delayed, and expression of β4 integrin and p21 was reduced in lysates of constitutive and wounded CLIC4(NULL) skin. The induced level of phosphorylated Smad2 in response to TGF-β was reduced in cultured CLIC4(NULL) keratinocytes relative to in wild-type cells, and CLIC4(NULL) keratinocytes migrated slower than did wild-type keratinocytes and did not increase migration in response to TGF-β. CLIC4(NULL) keratinocytes were also less adherent on plates coated with matrix secreted by wild-type keratinocytes. These results indicate that CLIC4 participates in skin healing and corneal wound reepithelialization through enhancement of epithelial migration by a mechanism that may involve a compromised TGF-β pathway.     

Adenosine promotes wound healing and mediates angiogenesis in response to tissue injury via occupancy of A(2A) receptors

Recent evidence indicates that topical application of adenosine A(2A) receptor agonists, unlike growth factors, increases the rate at which wounds close in normal animals and promotes wound healing in diabetic animals as well as growth factors, yet neither the specific adenosine receptor involved nor the mechanism(s) by which adenosine receptor occupancy promotes wound healing have been fully established. To determine which adenosine receptor is involved and whether adenosine receptor-mediated stimulation of angiogenesis plays a role in promotion of wound closure we compared the effect of topical application of the adenosine receptor agonist CGS-21680 (2-p-[2-carboxyethyl]phenethyl-amino-5'-N-ethylcarboxamido-adenosine) on wound closure and angiogenesis in adenosine A(2A) receptor knockout mice and their wild-type littermates. There was no change in the rate of wound closure in the A(2A) receptor knockout mice compared to their wild-type littermates although granulation tissue formation was nonhomogeneous and there seemed to be greater inflammation at the base of the wound. Topical application of CGS-21680 increased the rate of wound closure and increased the number of microvessels in the wounds of wild-type mice but did not affect the rate of wound closure in A(2A) receptor knockout mice. Similarly, in a model of internal trauma and repair (murine air pouch model), endogenously produced adenosine released into areas of internal tissue injury stimulates angiogenesis because there was a marked reduction in blood vessels in the walls of healing air pouches of A(2A) receptor knockout mice compared to their wild-type controls. Inflammatory vascular leakage and leukocyte accumulation in the inflamed air pouch were similarly reduced in the A(2A) receptor knockout mice reflecting the reduced vascularity. Thus, targeting the adenosine A(2A) receptor is a novel approach to promoting wound healing and angiogenesis in normal individuals and those suffering from chronic wounds.     

Impact of Mucorales and Other Invasive Molds on Clinical Outcomes of Polymicrobial Traumatic Wound Infections

Combat trauma wounds with invasive fungal infections (IFIs) are often polymicrobial with fungal and bacterial growth, but the impact of the wound microbiology on clinical outcomes is uncertain. Our objectives were to compare the microbiological features between IFI and non-IFI wounds and evaluate whether clinical outcomes differed among IFI wounds based upon mold type. Data from U.S. military personnel injured in Afghanistan with IFI wounds were examined. Controls were matched by the pattern/severity of injury, including blood transfusion requirements. Wound closure timing was compared between IFI and non-IFI control wounds (with/without bacterial infections). IFI wound closure was also assessed according to mold species isolation. Eighty-two IFI wounds and 136 non-IFI wounds (63 with skin and soft tissue infections [SSTIs] and 73 without) were examined. The time to wound closure was longer for the IFI wounds (median, 16 days) than for the non-IFI controls with/without SSTIs (medians, 12 and 9 days, respectively; P < 0.001). The growth of multidrug-resistant Gram-negative rods was reported among 35% and 41% of the IFI and non-IFI wounds with SSTIs, respectively. Among the IFI wounds, times to wound closure were significantly longer for wounds with Mucorales growth than for wounds with non-Mucorales growth (median, 17 days versus 13 days; P < 0.01). When wounds with Mucorales and Aspergillus spp. growth were compared, there was no significant difference in wound closure timing. Trauma wounds with SSTIs were often polymicrobial, yet the presence of invasive molds (predominant types: order Mucorales, Aspergillus spp., and Fusarium spp.) significantly prolonged the time to wound closure. Overall, the times to wound closure were longest for the IFI wounds with Mucorales growth.     

Engineering bone regeneration with bioabsorbable scaffolds with novel microarchitecture

Critical-sized defects (CSDs) were introduced into rat calvaria to test the hypothesis that absorption of surrounding blood, marrow, and fluid from the osseous wound into a bioabsorbable polymer matrix with unique microarchitecture can induce bone formation via hematoma stabilization. Scaffolds with 90% porosity, specific surface areas of approximately 10 m2/g, and median pore sizes of 16 and 32 microm, respectively, were fabricated using an emulsion freeze-drying process. Contact radiography and radiomorphometry revealed the size of the initial defects (50 mm2) were reduced to 27 +/- 11 mm2 and 34 +/- 17 mm2 for CSDs treated with poly(D,L-lactide-co-glycolide). Histology and histomorphometry revealed scaffolds filled with significantly more de novo bone than negative controls (p < 0. 007), more osteoid than both the negative and autograft controls (p < 0.002), and small masses of mineralized tissue (< 15 mm in diameter) observed within the scaffolds. Based on these findings, we propose a change in the current paradigm regarding the microarchitecture of scaffolds for in vivo bone regeneration to include mechanisms based on hematoma stabilization.     

Evaluation of fibrin sealants in cutaneous wound closure.

Human fibrin sealant (HFS) and bovine fibrin sealant (BFS) were delivered as preformulated fibrinogen-thrombin mixtures that are light activated. These formulations were evaluated in the healing of incised cutaneous wounds in beagle dogs. Four groups were differentiated by sealant type and study duration with group: BFS for 10 days, HFS for 10 days, BFS for 30 days, and HFS for 30 days. Healing was evaluated by noting incidences of open wounds, laser Doppler perfusion imaging (LDPI), planimetry, breaking strength, and histopathology. In the absence of tension, both sealants tended to hold wound edges together; however, HFS tended to be better than its controls and BFS. Both sealants augmented suture closure, necessitating fewer sutures for wound closure. At 5 and 30 days BFS wounds had more perfusion than HFS wounds, indicating more inflammation. At 10 and 30 days BFS wounds had larger scar areas than their controls, while scar areas of HFS wounds were smaller than either BFS wounds or controls. Breaking strengths indicated that HFS wounds were stronger than their controls and BFS wounds. Histologically, mild to moderate chronic-active inflammation was observed in wounds receiving either sealant, and this persisted longer in BFS wounds. Overall, HFS had positive qualities, thus showing potential for functional and cosmetic wound closure.     

Wound closure with EDC cross-linked cultured skin substitutes grafted to athymic mice

Collagen-glycosaminoglycan (C-GAG) sponges are commonly utilized as a substitute for the extracellular matrix of dermal tissue. Cultured skin substitutes (CSS) were assessed, after fabrication using sponges cross-linked with 1-ethyl-3-3-dimethylaminopropylcarbodiimide hydrochloride (EDC) at 0, 1, 5, or 50 mm, for development of viable, stratified skin tissue anatomy in vitro, and for wound contraction and cell viability in vivo. Cross-linking the C-GAG sponges with EDC reduced in vitro contraction of the CSS from a 39% reduction in area in the 0 mm CSS to 0% in the 50 mm group. Conversely, the wounds closed with 0, 1 and 5 mm EDC groups exhibited significantly less wound contraction than the 50 mm group. Engraftment of human cells occurred in 86%, 83%, and 83% of the wounds treated with CSS fabricated using 0, 1, and 5 mm EDC cross-linked sponges, respectively, which were significantly higher engraftment rates than the 50 mm group (17%). These data suggest that low concentrations of EDC can be used to improve the biochemical stability of the C-GAG component of CSS in vitro, and promote stable wound closure.     

ADAM12: a potential target for the treatment of chronic wounds

Wound healing is a complex process involving multiple cellular events, including cell proliferation, migration, and tissue remodeling. A disintegrin and metalloprotease 12 (ADAM12) is a membrane-anchored metalloprotease, which has been implicated in activation-inactivation of growth factors that play an important role in wound healing, including heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) and insulin growth factor (IGF) binding proteins. Here, we report that expression of ADAM12 is fivefold upregulated in the nonhealing edge of chronic ulcers compared to healthy skin, based on microarrays of biopsies taken from five patients and from healthy controls (p = 0.013). The increase in ADAM12 expression in chronic ulcers was confirmed by quantitative real-time polymerase chain reaction (RT-PCR). Moreover, immunohistochemical analysis demonstrated a pronounced increase in the membranous and intracellular signal for ADAM12 in the epidermis of chronic wounds compared to healthy skin. These findings, coupled with our previous observations that lack of keratinocyte migration contributes to the pathogenesis of chronic ulcers, prompted us to evaluate how the absence of ADAM12 affects the migration of mouse keratinocytes. Skin explants from newborn ADAM12-/- or wild-type (WT) mice were used to quantify keratinocyte migration out of the explants over a period of 7 days. We found a statistically significant increase in the migration of ADAM12-/- keratinocytes compared to WT control (p = 0.0014) samples. Taken together, the upregulation of ADAM12 in chronic wounds and the increased migration of keratinocytes in the absence of ADAM12 suggest that ADAM12 is an important mediator of wound healing. We hypothesize that increased expression of ADAM12 in chronic wounds impairs wound healing through the inhibition of keratinocyte migration and that topical ADAM12 inhibitors may therefore prove useful for the treatment of chronic wounds.     

Influence of electrospun collagen on wound contraction of engineered skin substitutes

The treatment of massive full-thickness burns with engineered skin substitutes has shown promise in clinical trials. The majority of skin substitutes are comprised of fibroblasts and/or keratinocytes on collagen scaffolds, commonly generated by freeze drying which can generate significant structural heterogeneity. Electrospinning may generate collagen scaffolds with greater homogeneity. Skin substitutes were fabricated using either freeze-dried (FD) or electrospun (ES) collagen scaffolds. Cell distribution, proliferation, organization, and maturation were assessed on each scaffold type in vitro, and engraftment and healing of full thickness wounds in athymic mice were tested. In vitro evaluation of freeze-dried collagen skin substitutes (FCSS) and electrospun collagen skin substitutes (ECSS) revealed no significant differences in cell proliferation, surface hydration, or cellular organization between the ECSS and FCSS groups. Both groups exhibited excellent stratification with a continuous layer of basal keratinocytes present at the dermal-epidermal junction. After grafting to full thickness wounds in athymic mice, both skin substitutes had high rates of engraftment: 87.5% in the FCSS group and 100% in the ECSS group. Histological evaluation of wounds revealed that bovine collagen persisted in the wound at week 8 in the FCSS group while no bovine collagen was seen in the ECSS group. At 8 weeks post-grafting, the ECSS grafts were 61.3+/-7.9% original graft area whereas the FCSS grafts were 39.2+/-8.8% original area (p<0.01). These results indicate that ES scaffolds can be used to fabricate skin substitutes with optimal cellular organization and can potentially reduce wound contraction compared to FD scaffolds. These advantages may lead to reduced morbidity in patients treated with skin substitutes fabricated from ES collagen.     

Cross-linked hyaluronate hydrogel prevents adhesion formation and reformation in mouse uterine horn model

The aim of this study was to evaluate the efficacy of cross-linked hyaluronate hydrogel (HA gel) as an adjuvant for postoperative adhesion prevention, in a mouse uterine horn model. In experiment 1 uterine horns were abrased with iodine. HA gel was applied to the injured surface before closure in the treatment group. In experiment 2, after injuring the uterine horns, three stitches were placed at equal distances around the uterine horns to appose the injured medial surfaces of the two horns during healing. HA gel was inserted between the uterine horns in the treatment group. In experiment 3 prevention of adhesion reformation was assessed. After lysis of adhesions that were induced as in experiment 2, HA gel was introduced between the serosal surfaces of apposing uterine horns. Untreated animals served as controls in each experiment. Statistical analysis was carried out using Student's t-test. The adhesion score was significantly lower in the HA gel group on the 14th day compared with controls in all the experiments: in experiment 1, 0.3 +/- 0.4 versus 1.7 +/- 1.2; in experiment 2, 0.9 +/- 1.0 versus 2.6 +/- 0.5; and in experiment 3, 1.5 +/- 0.9 versus 2.2 +/- 0.6 respectively. Cross-linked HA gel significantly reduced de-novo adhesions (P< 0.03) and adhesion reformation (P < 0.03).     

The antifibrogenic effects of liposome-encapsulated IFN-alpha2b cream on skin wounds.

Interferons (IFN), including IFN-alpha2b, have been used as antifibrogenic factors to modulate the excessive production of extracellular matrix (ECM) associated with dermal fibroproliferative disorders. This study was conducted to examine the ability of a dermal cream containing liposome-encapsulated IFN-alpha2b (LIPO+IFN) to affect the synthesis of ECM in open and reepithelialized wounds. Full-thickness skin wounds in 32 female Hartley guinea pigs (6 wounds per animal, 3 on each side) were made with an 8-mm biopsy punch. Each wound on the right side received 3,000 U LIPO+IFN, whereas wounds on the left side received cream containing empty liposomes. Histologic examination revealed a significant reduction in scar formation in LIPO+IFN-treated but not in vehicle-treated wounds. Northern analysis showed reductions in type I procollagen mRNA in healed wounds treated with LIPO+IFN (day 4 groups: 1596.9 +/- 207 vs. 3710.2 +/- 493 densitometry units, p < 0.01, n = 8). This was consistent with a reduction in the concentration of collagen in the tissue, assayed as 4-hydroxyproline (day 4 group: 38.5 +/- 3.8 vs. 54.5 +/- 3.9 microg per tissue, p < 0.01, n = 8). Even when applied to reepithelialized wounds, LIPO+IFN caused a marked reduction in type I collagen mRNA (1938.5 +/- 579 vs. 4085.7 +/- 1271 densitometry units, p < 0.01, n = 8). These findings support the concept of the early topical use of this antifibrogenic agent for treatment of dermal fibroproliferative disorders, such as hypertrophic scars.     

Improvement of Schwann cell attachment and proliferation on modified hyaluronic acid strands by polylysine

Hyaluronic acid (HyA) has the intrinsic ability to promote cell proliferation and reduce scar formation. However, the clinical use of HyA has so far been limited because of its water solubility and nonadhesive characteristics. Increasing interest in HyA as a clinically useful biomaterial has prompted our study of altering HyA's physical properties to render it a potential component of nerve grafts. In this study, strands of HyA were cross-linked by glutaraldehyde (Glut), coated with polylysine, and then inoculated with Schwann cells (SCs). Results in vivo and in vitro demonstrated that cross-linked HyA strands were water insoluble and thus less biodegradable. Poly-D-lysine-resurfaced strands showed significant SC attachment of 350-400 cells/mm(2), compared to uncoated controls (0-10 cells/mm(2), p < 0.01). Fibroblast control groups showed an attachment of 40-100 cells/mm(2) on coated strands. Immunostaining for proliferating cells showed SCs as and fibroblasts as +. Cells neither adhered to nor proliferated on the modified HyA strands that were not resurfaced. The results suggest that polylysine promotes SC attachment and proliferation to glutaraldehyde-cross-linked HyA strands, the product being a three-dimensional composite with low solubility that may have potential application in nerve grafts.     

Diminished type III collagen promotes myofibroblast differentiation and increases scar deposition in cutaneous wound healing

The repair of cutaneous wounds in the postnatal animal is associated with the development of scar tissue. Directing cell activities to efficiently heal wounds while minimizing the development of scar tissue is a major goal of wound management and the focus of intensive research efforts. Type III collagen (Col3), expressed in early granulation tissue, has been proposed to play a prominent role in cutaneous wound repair, although little is known about its role in this process. To establish the role of Col3 in cutaneous wound repair, we examined the healing of excisional wounds in a previously described murine model of Col3 deficiency. Col3 deficiency (Col3+/-) in aged mice resulted in accelerated wound closure with increased wound contraction. In addition, Col3-deficient mice had increased myofibroblast density in the wound granulation tissue as evidenced by an increased expression of the myofibroblast marker, α-smooth muscle actin. In vitro, dermal fibroblasts obtained from Col3-deficient embryos (Col3+/- and -/-) were more efficient at collagen gel contraction and also displayed increased myofibroblast differentiation compared to those harvested from wild-type (Col3+/+) embryos. Finally, wounds from Col3-deficient mice also had significantly more scar tissue area on day 21 post-wounding compared to wild-type mice. The effect of Col3 expression on myofibroblast differentiation and scar formation in this model suggests a previously undefined role for this ECM protein in tissue regeneration and repair.     

Low-Level Laser Therapy Facilitates Superficial Wound Healing in Humans: A Triple-Blind, Sham-Controlled Study

OBJECTIVE: Low-level laser therapy (LLLT) has been promoted for its beneficial effects on tissue healing and pain relief. However, according to the results of in vivo studies, the effectiveness of this modality varies. Our purpose was to assess the putative effects of LLLT on healing using an experimental wound model. DESIGN AND SETTING: We used a randomized, triple-blind, placebo-controlled design with 2 within-subjects factors (wound and time) and 1 between-subjects factor (group). Data were collected in the laboratory setting. SUBJECTS: Twenty-two healthy subjects (age = 21 +/- 1 years, height = 175.6 +/- 9.8 cm, mass = 76.2 +/- 14.2 kg). MEASUREMENTS: Two standardized 1.27-cm(2) abrasions were induced on the anterior forearm. After wound cleaning, standardized digital photos were recorded. Each subject then received LLLT (8 J/cm(2); treatment time = 2 minutes, 5 seconds; pulse rate = 700 Hz) to 1 of the 2 randomly chosen wounds from either a laser or a sham 46-diode cluster head. Subjects reported back to the laboratory on days 2 to 10 to be photographed and receive LLLT and on day 20 to be photographed. Data were analyzed for wound contraction (area), color changes (chromatic red), and luminance. RESULTS: A group x wound x time interaction was detected for area measurements. At days 6, 8, and 10, follow-up testing revealed that the laser group had smaller wounds than the sham group for both the treated and the untreated wounds (P < .05). No group x wound x time differences were detected for chromatic red or luminance. CONCLUSIONS: The LLLT resulted in enhanced healing as measured by wound contraction. The untreated wounds in subjects treated with LLLT contracted more than the wounds in the sham group, so LLLT may produce an indirect healing effect on surrounding tissues. These data indicate that LLLT is an effective modality to facilitate wound contraction of partial-thickness wounds.     

Wound contraction is significantly reduced by the use of microcarriers to deliver keratinocytes and fibroblasts in an in vivo pig model of wound repair and regeneration

In full-thickness injuries caused by extensive burns or penetrating traumatic injuries, the natural epidermal stem cell niche is destroyed, and wound healing occurs through migration of cells from the wound edges and wound contraction. This can lead to significant contracture formation, especially in large full-thickness injuries, causing lack of mobility and pain. Contraction is reduced when wounds are treated using split-thickness skin grafts (STSG) or dermal substitutes, particularly in combination with cultured autologous keratinocytes, delivered as confluent sheets or sprayed as a single cell suspension (SAK). Here, we show that the application of keratinocytes alone or keratinocytes with fibroblasts, delivered on microcarriers, in combination with STSG or a dermal substitute, significantly reduces contraction of wounds in vivo in a porcine model of wound repair and regeneration. A decrease in alpha-smooth muscle actin-positive myofibroblasts, the cell type responsible for wound contraction, accompanies the reduction in contraction. These findings demonstrate the potential for a significant clinical advantage in the treatment of full-thickness injuries.     

Beta-tricalcium phosphate plugs for press-fit fixation in ACL reconstruction--a mechanical analysis in bovine bone

BACKGROUND: The goal of this study was to test fixation properties of microporous pure beta-tricalcium phosphate (TCP) plugs (porosity 40%) for press-fit fixation of the ACL graft using patellar tendons with and without bone blocks. We set out to establish whether it is possible, in this way, to obtain results comparable with those of interference screw fixation of bone-tendon-bone (BTB) grafts in terms of cyclic loading and load-to-failure. METHODS: In a bovine model 30 ACL grafts were fixed in tibial drill holes, divided into three groups: 10 BTB grafts fixed with TCP press-fit plugs (7x25 mm), 10 pure patellar tendon grafts with TCP press-fit plugs (7x25 mm), and 10 BTB grafts with metal interference screws (7x25 mm). All grafts were tested by cyclic loading (50-200 N) and loaded until failure in a tensiometer. RESULTS: Under cyclic loading one interference screw fixation failed. None of the TCP plug fixations failed. After 1500 cycles the displacement of the graft in the drill hole for BTB fixed with screws was 3.6+/-7.8 mm, for BTB/TCP plugs 1.6+/-3.4 mm, and for the pure tendon/TCP grafts 1.4+/-0.4 mm. Regarding cyclic loading the pure tendon/TCP system was significantly superior to BTB (p=0.007). The load-to-failure for the BTB/interference screw group was 908+/-539 N with a stiffness of 94+/-36 N/mm, 936+/-245 N for the BTB/TCP cylinder group with a stiffness of 98+/-12 N/mm, and 673+/-159 N for the pure tendon/TCP group with a stiffness of 117+/-9 N/mm. In terms of pull-out load the BTB/TCP system was significantly better than the pure tendon/TCP group (p=0.011). However, pure tendon/TCP grafts achieved significantly greater stiffness (p=0.002) than the BTB system. CONCLUSION: Press-fit fixation with microporous pure beta-TCP plugs of BTB grafts or patellar tendon grafts without bone blocks for ACL reconstruction leads to primary stability comparable with that achieved by fixation with metal interference screws in case of BTB grafts.     

Epidermis promotes dermal fibrosis: role in the pathogenesis of hypertrophic scars

Hypertrophic scarring is a pathological process characterized by fibroblastic hyperproliferation and by excessive deposition of extracellular matrix components. It has been hypothesized that abnormalities in epidermal-dermal crosstalk explain this pathology. To test this hypothesis, a tissue-engineered model of self-assembled reconstructed skin was used in this study to mimic interactions between dermal and epidermal cells in normal or pathological skin. These skin equivalents were constructed using three dermal cell types: normal wound (Wmyo) or hypertrophic wound (Hmyo) myofibroblasts and normal skin fibroblasts (Fb). Epidermis was reconstructed with normal skin keratinocytes (NK) or hypertrophic scar keratinocytes (HK). In the absence of keratinocytes, Hmyo formed a thicker dermis than Wmyo. When seeded with NK, the dermal thickness of Hmyo (121.2 +/- 31.4 microm vs 196.2 +/- 27.8 microm) and Fb (43.7 +/- 7.1 microm vs 83.6 +/- 16.3 microm) dermis was significantly (p < 0.05) reduced, while that of Wmyo (201.5 +/- 15.7 microm vs 160.7 +/- 21.1 microm) was increased. However, the presence of HK always induced significantly thicker dermis formation than observed with NK (Wmyo: 238.8 +/- 25.9 microm; Hmyo: 145.5 +/- 22.4 microm; Fb: 74.2 +/- 11.2 microm). These results correlated with collagen and MMP-1 secretion and with cell proliferation, which were increased when keratinocytes were added, except for the collagen secretion of Hmyo and Fb in the presence of NK. The level of dermal apoptosis was not different when epidermis was added to the dermis (<1% in each category). These observations strongly suggest that hypertrophic scar keratinocytes play a role in the development of pathological fibrosis by influencing the behaviour of dermal cells.     

Erythropoietin improves skin wound healing and activates the TGF-β signaling pathway

We could recently report that erythropoietin (EPO) accelerates skin wound healing in mice. Now, we provide insight into the molecular mechanisms of this non-hematopoietic property of EPO analyzing the transforming growth factor (TGF)-β signaling pathway. EPO receptor was found expressed in both non-wounded and wounded skin tissue as well as in fibroblasts and keratinocytes. In saline-treated control animals, wounds exhibited a significant upregulation of TGF-β1 and of α-smooth muscle actin (α-SMA) compared with non-wounded skin. EPO treatment accelerated wound epithelialization and induced mRNA expression of TGF-β1 and α-SMA. In addition, EPO significantly enhanced phosphorylation of Smad2 and Smad3 in fibroblasts and also elevated phosphorylation of Smad3 in wound tissue. Blockade of TGF-β using a neutralizing anti-TGF-β antibody attenuated EPO-induced acceleration of wound epithelialization in vivo and markedly reversed EPO effects on mRNA expression of TGF-β1 and α-SMA. In conclusion, EPO caused activation of the Smad-dependent TGF-β signaling pathway, enhanced differentiation of myofibroblasts, and accelerated skin wound closure.     

An in vivo study of hydrogels based on physiologically clotted fibrin-gelatin composites as wound-dressing materials

In this study we have reported the efficacy of three biomaterials: (a) physiologically clotted fibrin-gelatin composite (PFG), (b) PFG graft copolymerized with 2-hydroxyethyl methacrylate (PFG-HEMA), and (c) PFG graft copolymerized with 2-hydroxypropyl methacrylate (PFG-HPMA) as temporary wound-dressing materials using the rat as an animal model. Full-thickness excision wounds were made on the back of female rats weighing about 150 +/- 10 g. The dressings were applied on the wounds and changed periodically at an interval of 4 days with the respective materials. The wounds treated with PFG-HEMA healed completely on 15th day after wound creation, whereas those treated with PFG and PFG-HPMA resulted in complete healing on the 17th day. The concentrations of collagen, hexosamine, and uronic acid in the granulation tissue were determined. The PFG and its graft copolymers acted as hydrogels, thereby absorbing excess exudates, while still maintaining a moist environment at the wound site. The enhanced wound healing in the experimental animals was reflected in the increased rate of wound contraction. The results of the histological and mechanical studies of the experimental groups revealed that reepithelialization and remodeling of the skin have been achieved by providing a moist environment at the wound site by the biomaterials and thereby hastening the migration of keratinocytes.     

Effects of auricular chondrocyte expansion on neocartilage formation in photocrosslinked hyaluronic acid networks

The overall objective of this study was to examine the effects of in vitro expansion on neocartilage formation by auricular chondrocytes photoencapsulated in a hyaluronic acid (HA) hydrogel as a next step toward the clinical application of tissue engineering therapies for treatment of damaged cartilage. Swine auricular chondrocytes were encapsulated either directly after isolation (p = 0), or after further in vitro expansion ( p = 1 and p = 2) in a 2 wt%, 50-kDa HA hydrogel and implanted subcutaneously in the dorsum of nude mice. After 12 weeks, constructs were explanted for mechanical testing and biochemical and immunohistochemical analysis and compared to controls of HA gels alone and native cartilage. The compressive equilibrium moduli of the p = 0 and p = 1 constructs (51.2 +/- 8.0 and 72.5 +/- 35.2 kPa, respectively) were greater than the p = 2 constructs (26.8 +/- 14.9 kPa) and the control HA gel alone (12.3 +/- 1.3 kPa) and comparable to auricular cartilage (35.1 +/- 12.2 kPa). Biochemical analysis showed a general decrease in glycosaminoglycan (GAG), collagen, and elastin content with chondrocyte passage, though no significant differences were found between the p = 0 and p = 1 constructs for any of the analyses. Histological staining showed intense and uniform staining for aggrecan, as well as greater type II collagen versus type I collagen staining in all constructs. Overall, this study illustrates that constructs with the p = 0 and p = 1 auricular chondrocytes produced neocartilage tissue that resembled native auricular cartilage after 12 weeks in vivo. However, these results indicate that further expansion of the chondrocytes (p = 2) can lead to compromised tissue properties.     

Disruption of Interleukin-1 Signaling Improves the Quality of Wound Healing

In this study, we investigated the role of interleukin (IL)-1 signaling in wound healing. IL-1 receptor type I (IL-1R) knockout (KO) mice showed reduced fibrosis in both cutaneous and deep tissue wounds, which was accompanied by a reduction in inflammatory cellular infiltration in cutaneous but not in deep tissue wounds. There were no differences in either total collagenolytic activity or in the expression of selected matrix metalloproteinases or tissue inhibitors of metalloproteinases between the wound fluids from wild-type or IL-1R KO mice. However, wound fluids from IL-1R KO mice contained lower levels of IL-6 compared with wild-type controls. In addition, the infusion of IL-6 into wounds in IL-1R KO mice did not increase fibrosis. Skin wounds in IL-1R KO animals had lower levels of collagen and improved restoration of normal skin architecture compared with skin wounds in wild-type mice. However, neither the tensile strength of incisional skin wounds nor the rate of closure of excisional wounds differed between IL-1R KO and wild-type animals. The reduced fibrotic response in wounds from IL-1R KO mice could be reproduced by the administration of an IL-1R antagonist. These findings suggest that pharmacological interference with IL-1 signaling could have therapeutic value in the prevention of hypertrophic scarring and in the treatment of fibrotic diseases.Progress in the therapeutic management of abnormal wound healing has fallen short of expectations. The promise of molecular medicine to normalize impaired healing, as seen in diabetes, vascular insufficiency, or other chronic diseases, through the use of exogenous cytokines or growth factors has not been realized. At the other end of the abnormal wound healing spectrum, no reliable prophylactic or therapeutic measures exist to address the pathologies of excessive repair, exemplified by hypertrophic burn scars, keloids, and stenosing gastrointestinal or vascular anastomoses. The availability of effective therapies that allow for the modulation of the wound healing response would be of substantial clinical relevance. Recent reports demonstrate a markedly reduced cellular inflammatory response in models of sterile inflammation^1,2,3,4 and decreased scarring after experimental myocardial infarction in mice deficient in the interleukin (IL)-1 receptor type I (IL-1R).⁴The present studies tested the hypothesis that genetic or pharmacological interference with IL-1 signaling would modulate the inflammatory response in skin and deep tissue wounds and reduce scar formation. Results using IL-1R knockout (KO) mice demonstrated that signaling through the IL-1R is required for the constitution of a normal cellular inflammatory response in cutaneous but not in deep tissue wounds. Most importantly, the quality of wound healing was different in IL-1R KOs, with cutaneous wounds in these animals attaining better restoration of normal skin architecture and a marked reduction in fibrosis without compromise in tensile strength. Additionally, deep tissue wounds in IL-1R KO mice showed a substantial reduction in collagen content, an observation that was reproduced by the administration of a human recombinant IL-1 R antagonist.Findings demonstrate a role for the IL-1/IL-1R axis in the regulation of wound healing. They suggest that interference with IL-1 signaling through the use of an IL-1R antagonist may find a clinical application in the prevention of excessive or hypertrophic scar formation.