Accelerated diabetic wound healing using cultured dermal fibroblasts retrovirally transduced with the platelet-derived growth factor B gene

The treatment of diabetic wounds is a considerable clinical challenge. In this study, mouse dermal fibroblasts retrovirally transduced with the human platelet-derived growth factor B (PDGF-B) gene were used to treat diabetic mouse wounds. The PDGF-B gene was obtained from human umbilical vein endothelial cells, cloned into retroviral vectors, and introduced into diabetic mouse C57B1/ks-db/db dermal fibroblasts. In vitro results demonstrated production of PDGF-B protein by these transduced cells at steady-state levels of 1000 ng PDGF-B/10(6) cells/24 hours, and expression of PDGF-B mRNA. These cells were seeded onto polyglycolic acid scaffold matrices and used to treat diabetic mouse 20-mm x 20-mm full-thickness excisional dorsal skin wounds. Measurement of the residual epithelial gap at 21 days showed significantly accelerated healing (P < 0.05) of wounds treated with PDGF-transduced cells (epithelial gap 10.46 +/- 1.20 mm) compared with untreated wounds (14.66 +/- 0.591 mm), wounds treated with polyglycolic acid alone (14.80 +/- 0.575 mm), or wounds treated with negative control LNCX-transduced cells (13.76 +/- 0.831 mm). Immunohistochemical staining showed intense staining for PDGF in wounds treated with PDGF-B-transduced cells. This study demonstrates the promising potential for gene therapy in diabetic wound healing.     

Lentiviral gene transfer of SDF-1alpha to wounds improves diabetic wound healing

BACKGROUND: Chronic wounds continue to be a major clinical problem and novel therapeutic approaches are needed. We have previously demonstrated that treatment of diabetic mouse wounds with local application of stromal progenitor cells results in improved healing and increased production of stromal-derived growth factor-1alpha (SDF-1alpha). We hypothesized that lentiviral-mediated increased production of SDF-1alpha in the wound environment could also improve diabetic wound healing. MATERIALS AND METHODS: Full-thickness excisional wounds were created in Db-/Db- mice and immediately treated with 10(6), 10(8), or 10(9) plaque-forming units of a lentiviral construct containing GFP-SDF-1alpha or GFP alone. At 7 and 14 days post wounding, wounds were harvested for histological and molecular analysis. RESULTS: At 7 days, Db-/Db- wounds treated with lenti GFP-SDF-1alpha exhibited a decrease in wound surface area for all doses tested. Morphologically, SDF-treated wounds were more cellular with increased granulation tissue volume compared to controls (P < 0.05). GFP expression was maintained in treated tissue at 7 days post wounding, but little expression was observed at 14 days. While we did not observe a difference in the gross wound surface area at 14 days, histological analysis revealed that SDF-treated wounds were fully epithelialized (n = 6) compared to only one of six controls. CONCLUSIONS: Lentiviral-mediated overproduction of SDF-1alpha is sufficient to correct the pathophysiologic abnormalities in diabetic wound healing resulting in complete epithelialization at 2 weeks. SDF-1alpha-mediated improvement in diabetic wound healing has significant implications for the development of novel therapeutic strategies to facilitate wound closure which target progenitor cell mobilization and recruitment.     

Fibrin sealant combined with fibroblasts and platelet-derived growth factor enhance wound healing in excisional wounds

We test the hypothesis that the fibrinogen-thrombin formulation of fibrin sealant combined with fibroblasts and PDGF-BB enhance cutaneous wound healing. Four formulations varying in fibrinogen and thrombin concentration were applied to full-thickness biopsy wounds in the rabbit ear cutaneous wound healing model with or without cultured rabbit dermal fibroblasts (RDFs; 3 × 10⁵ cells/wound) embedded in the fibrinogen component. At post-wounding day 7, there was no difference in the diluted vs. non-diluted formulations for either the promotion of granulation tissue coverage of the open wounds or total granulation tissue area when tested without embedded cells. Including the RDFs, the highest degree of wound coverage by granulation tissue was observed in the combined dilution formulation (17.3 mg/mL fibrinogen, 167 U/mL thrombin; n =10 wounds) that was 167% ( p <0.05) of the nondiluted FS containing cells (50 mg/mL fibrinogen, 250 U/mL thrombin; n =10 wounds). Inclusion of fibroblasts increased granulation tissue area within the wounds vs. FS alone ( p <0.05) for each diluted formulation although no differences in this parameter were observed within each group (FS alone or with embedded cells). However, addition of the vulnerary growth factor PDGF-BB (3 mg; n =4) with the embedded RDFs in the combined dilution formulation increased granulation tissue area over two-fold ( p <0.01) over FS alone. Additionally, the presence of the RDFs promoted incorporation of the granulation tissue with and epithelial migration over the FS suggesting an active interaction between cells delivered to the wound by FS and the host repair cells. The findings suggest the progress of cutaneous defect repair can be enhanced by ex vivo cell delivery in fibrin sealant.     

Locally applied platelet-derived growth factor accelerates fracture healing

Platelet-derived growth factor (PDGF) is known to stimulate osteoblast or osteoprogenitor cell activity. We investigated the effect of locally applied PDGF from poly-D,L-lactide (PDLLA)-coated implants on fracture healing in a rat model. A closed fracture of the right tibia of four-month-old Sprague-Dawley rats (n = 40) was stabilised with implants coated with a biodegradable PDLLA versus implants coated with PDLLA and PDGF. Radiographs were taken throughout the study, and a marker of DNA activity, bromodeoxyuridine (BrdU), was injected before the rats were killed at three, seven and ten days. The radiographs showed consolidation of the callus in the PDGF-treated group compared with the control group at all three time points. In the PDGF-treated group, immunohistochemical staining of BrdU showed that the distribution of proliferating cells in all cellular events was higher after ten days compared with that at three and seven days. These results indicate that local application of PDGF from biodegradable PDLLA-coated implants significantly accelerates fracture healing in experimental animals. Further development may help fracture healing in the clinical situation.     

rhPDGF对糖尿病鼠全层皮肤缺损创面微血管形成的影响和意义

目的:观察外源性重组人血小板源性生长因子(rhPDGF)对全层皮肤缺损的糖尿病鼠创面愈合过程中微血管形成的影响及促创面愈合的作用。方法:利用大鼠全层皮肤缺损创面愈合模型,将26只非糖尿病鼠52个创面分为3组:①A组为糖尿病大鼠创伤自然愈合组;②B组为接受rhPDGF治疗的糖尿病大鼠创伤愈合组;③C组为赋性剂组。于伤后3 d、7 d和14 d采取创面皮肤标本,用组织病理HE和血管Ⅷ因子抗体免疫组织化学染色技术检测创面肉芽再生与再血管化情况进行观察,同时观察创面的愈合情况。结果:全层皮肤缺损糖尿病鼠创面肉芽组织中微血管的形成数量少,采用赋性剂对照组与糖尿病鼠自然愈合组无显著差别。但外源性使用rhPDGF后,肉芽组织的形成量明显增多,内含的微小血管数量显著增加,愈合能力明显增强。结论:糖尿病大鼠微血管形成障碍可能是其创面愈合延迟的重要因素。而应用外源性的PDGF有助于微血管的形成,可以改善糖尿病鼠的创面愈合能力。     

脂质体介导血小板衍化生长因子-B基因转染成纤维细胞的表达

目的用脂质体介导血小板衍化生长因子(PDGF)-B真核表达质粒转染成纤维细胞，使PDGF-B基因在成纤维细胞中特异表达。方法构建PDGF—B真核表达质粒，用脂质体LipofectAMINE介导转染成纤维细胞，G418筛选阳性克隆。逆转录-聚合酶链反应(RT-PCR)和细胞爬片免疫组织化学染色检测PDGF-B基因在成纤维细胞中的表达。结果RT—PCR检测结果显示，转染组PDGF-BmRNA的表达量较未转染组明显增加。免疫组织化学染色结果显示转染组成纤维细胞胞浆中有大量棕黄色阳性颗粒。结论脂质体介导PDGF-B基因成功转染成纤维细胞并表达。为PDGF基因治疗奠定基础。     

Effect of platelet-derived growth factor on rabbit corneal wound healing

Human recombinant platelet-derived growth factor was evaluated with the use of wound healing models in New Zealand albino rabbits. The efficacy of the platelet-derived growth factor dimers, AA, AB, and BB, was determined in corneal reepithelialization and anterior keratectomy models which examined the healing response in the presence or absence of the basement membrane. All dimers increased the rate of wound healing in both models at 100 µg/ml when compared with control; however, the platelet-derived growth factor-BB isoform showed the most dramatic increase in both studies. The strength of the healing stroma after incision was evaluated by means of a tensile strength model. Histologic evaluation of the stromal wound area after 9 days of healing showed a marked increase in the number of keratocytes within the wound bed of the corneas treated with platelet-derived growth factor-BB when compared with control corneas. In addition, at 9 days, the epithelial plug was still present in the control corneas but had been extruded to the surface by the granulation tissue in the platelet-derived growth factor-BB—treated corneas. These results are indicative of a more advanced stage of healing in treated versus control wounds at 9 days after the operation. A 30% increase in corneal tensile strength versus control was noted after 21 days of healing. Finally, in an in vitro gel contraction assay, platelet-derived growth factor exhibited a dose-dependent effect on the contraction of fibroblasts for doses ranging from 0.01 to 10 ng/ml. These results indicate that platelet-derived growth factor is active in the corneal wound healing process.     

Combination of platelet-derived growth factor-BB and insulin-like growth factor-I is more effective than platelet-derived growth factor-BB alone in stimulating complete healing of full-thickness wounds in "older" diabetic mice

Platelet-derived growth factor and insulin-like growth factor-I have been shown to interact synergistically to enhance repair of skin wounds in normal healing swine. Platelet-derived growth factor alone has shown promise in treating human chronic ulcers. The objective of this study was to compare the wound healing effects of platelet-derived growth factor-BB alone with those of a combination of platelet-derived growth factor-BB and insulin-like growth factor-I in an improved model with the use of "older" animals with diabetes. Older diabetic (db/db) mice (>15 weeks of age) have less elevated insulin levels compared with young db/db mice. The serum insulin levels in the older animals is 1.0 to 2.5 times that of the nondiabetic animals, a similar increase to that which occurs in human patients with type II diabetes. Healing was evaluated in two studies involving a total of 104 animals. Treatment groups included the following: 4.0 microg/cm(2) of platelet-derived growth factor-BB, 40.0 microg/cm(2) of platelet-derived growth factor-BB, 4.0 microg/cm(2) of both platelet-derived growth factor-BB and insulin-like growth factor-I or vehicle. All growth factors were applied topically in a methylcellulose vehicle to full-thickness wounds every other day for 24 days. Efficacy end points were median and mean time to complete healing and rate of wound closure. The median time to complete healing for animals receiving the platelet-derived growth factor-BB/insulin-like growth factor-I combination was 38% and 33% faster (p < 0.001) than animals receiving 4.0 microg/cm(2) and 40.0 microg/cm(2) of platelet-derived growth factor-BB, respectively. The mean time to complete healing for platelet-derived growth factor/insulin-like growth factor-I treated animals was 31% and 29% faster (p < 0.001) than 4.0 microg/cm(2) and 40.0 microg/cm(2) platelet-derived growth factor-BB treated animals, respectively. Wounds treated with 4.0 microg/cm(2) platelet-derived growth factor-BB/insulin-like growth factor-I healed, on average, in 22 days compared with 31 days for 40.0 microg/cm(2) platelet-derived growth factor-BB alone and 38 days for vehicle. Also, platelet-derived growth factor-BB/insulin-like growth factor-I significantly improved the rate of wound closure throughout the duration of the studies compared with either dose of platelet-derived growth factor-BB alone (p < 0.005) or vehicle (p < 0.001). In conclusion, the data show that the combination of platelet-derived growth factor-BB and insulin-like growth factor-I is more effective than platelet-derived growth factor-BB alone at the doses tested or vehicle treatment in stimulating cutaneous wound healing in older, diabetic mice.     

Effects of mesenchymal stem cells transfected with human hepatocyte growth factor gene on healing of burn wounds

Objective： To explore the effects of bone marrow-derived mesenchymal stem cells （BMSCs）transfected with adenoviral vector carrying hepatocyte growth factor （HGF, Ad-HGF） on burn wound healing.Methods： BMSCs from male Wistar rats were separated and purified with Percoll separating medium by density gradient centrifugation and cultured with DMEM containing 20% fetal bovine serum （FBS）. Then BMSCs were transfected with Ad-HGF at the optimal gene transduction efficiency of 100 multiplicity of infection （MOI）. The efficiency of transfection and the expression of HGF in the suspension were detected by flow cytometry and enzyme linked immunosorbent assay （ELISA） respectively. Thirtytwo female rats were subjected to 90℃ water for 12 seconds to induce a partial thickness skin burn. The animals were randomly divided into mesenchymal stem cells （MSCs） treatment group （Group A）, Ad-HGF treatment group （Group B）,Ad-HGF-modified MSCs treatment group （Group C） and saline control group （Group D）. On days 3, 5, 7, 14 and 21 postburn, HE and Sirius red stain were performed to observe the burn wound healing and collagen content. The content of hydroxyproline in wounds was also detected.Transplanted cells and the expression of（sex-determining region Y） SRY gene were detected by in situ hybridization and polymerase chain reaction （PCR）, while the expression of HGF in wound tissues was detected by ELISA.Results： The result of flow cytometry showed that the transfection efficiency was 86.41% at 100 MOI. Compared with the control group, the content of HGF in the supernatant after transfection increased time-dependently and peaked at 48 h, showing significant differences at 24 h, 48 h,72 h and 96 h （P＜0.01 ）. Results of HE stain revealed that the range of re-epidermidalization in Group C was significantly larger than that in other groups in the first week. Three weeks postburn, the epidermis was significantly thicker in Group C than in other groups and the nails of dermis inserted into the derma of burn wounds. Sirius red stain showed that the content of collagen Ⅰ in Group C was much less compared with that in other groups 21 days postburn. In situ hybridization revealed an expression of SRY gene in burned female rats, consistent with the finding of PCR. Immunohistochemistry demonstrated the largest increase of HGF expression in Group C, whose contents of hydroxyproline,however, decreased on day 7 postburn. Compared with other groups, the content of HGF in the wounds of Group C increased obviously on day 14 after transfection （P＜0.05） and there was no significant difference among Groups A, B and D.Conclusion： Our study suggests that transplantation of MSCs modified with Ad-HGF has positive effects on the healing of burn wounds probably through differentiation and release of relevant cytokines.     

Wound healing potential of topical bacteriophage therapy on diabetic cutaneous wounds

Chronic wounds that fail to heal are a common complication of diabetes mellitus and the most common precipitating reason for nontraumatic lower limb amputation. Unfortunately, the bacterial species that cause these infections are becoming more resistant to antibiotics, making them increasingly difficult to treat. We assessed the feasibility of combating chronic bacterial infections with a topically delivered bacteriophage cocktail in two animal models of diabetes mellitus. Microbiological, planimetric, and histological parameters were compared in debrided infected wounds with or without topical bacteriophage treatment. We determined that bacteriophage treatment effectively decreased bacterial colony counts and improved wound healing, as indicated by smaller epithelial and dermal gaps, in Staphylococcus aureus and Pseudomonas aeruginosa infections but was not as effective against Acinetobacter baumannii. Although the improvements were more significant in the rodent model than in the porcine model, our results suggest that topically administered bacteriophage treatment may be effective in resolving chronic infections, especially when applied in conjunction with wound debridement. These findings have important implications for the feasibility of using topical antimicrobial therapies to safely treat chronic infections in diabetes mellitus patients.     

Influence of platelet-derived growth factor on microcirculation during normal and impaired wound healing

The aim of the current study was to evaluate the influence of platelet-derived growth factor (PDGF) on skin microcirculation during normal and impaired wound healing. Secondary healing wounds were created on the ears of hairless mice and treated once with 3 microg of PDGF-BB immediately after wound creation. Intravital fluorescence microscopy was used to quantify reepithelialization, revascularization, vessel diameters, vascular permeability, and leukocyte-endothelium interactions up to 24 days after wound creation. Microvascular perfusion was assessed by laser Doppler flowmetry. Wound healing was studied in normal (n = 15) and ischemic skin tissue (n = 15) as well as in mice (n = 17) rendered hyperglycemic by an intravenous injection of streptozotocin 7 days prior to wound creation. Treatment with PDGF accelerated reepithelialization and reduced the time for complete wound closure in ischemic skin from 14.9 +/- 2.5 (control) to 12.3 +/- 1.8 days (p < 0.03), and in hyperglycemic animals from 15.0 +/- 2.4 (control) to 12.0 +/- 3.0 days (p < 0.04). Revascularization of these wounds was also significantly enhanced after PDFG application. No other parameters were influenced by the treatment. Normal wound healing was not affected. This study confirms the positive influence of PDGF on wound healing under pathophysiological conditions. The effects in this model seem to be primarily due to the mitogenic potency of PDGF on keratinocytes and endothelial cells. A significant effect on leukocyte activation during the inflammatory process was not observed.     

Effect of transforming growth factor beta and basic fibroblast growth factor on steroid-impaired healing intestinal wounds.

A longitudinal intestinal wound model in the pig was used to assess the effect of parenteral steroids (betamethasone 12 mg 50 kg-1 intramuscularly twice daily) on breaking load. Steroid treatment significantly decreased the breaking load of wounds in the ileum and colon in comparison with wounds from saline-treated animals. In a further group of animals receiving steroids, paired longitudinal wounds were constructed. One wound of a pair was treated with a local application of transforming growth factor beta (TGF-beta) (5 micrograms per wound) or basic fibroblast growth factor (5 micrograms per wound) in a collagen suspension. The other wound was treated with a collagen suspension alone. Ileal wounds treated with TGF-beta were significantly stronger than collagen-treated controls at 7 days. The steroid-induced impairment of breaking load in intestinal wounds is partially reversed by a local application of TGF-beta in a collagen suspension at the time of surgery.     

促进骨外露创面愈合的研究

目的探讨肤疾骨宁膏联合表皮生长因子(EGF)促进骨外露创面的愈合作用。方法将45只家兔随机分为3组:(1)肤疾骨宁膏联合EGF组;(2)肤疾骨宁膏组;(3)生理盐水组。实验60d,观察创面愈合情况及应用免疫组织化学和原位杂交技术检测EGF/EGFR及其mRNA的表达。结果肤疾骨宁膏联合EGF组创面愈合最快,生理盐水组创面愈合最慢(P<0.01)。创面在应用肤疾骨宁膏后,EGF/EGFR及其mRNA表达量逐渐增高,EGF及其mRNA表达量在15d达到高峰,EGFR及其mRNA表达量高峰期为15～22d,较生理盐水组明显增高,并且高峰提前(P<0.01)。结论肤疾骨宁膏联合EGF可有效促进骨外露创面的愈合。     

血小板源性生长因子基因修饰的人工复合皮移植大鼠创面效果观察

目的观察血小板源性生长因子B(PDGFB)基因修饰的人工复合皮移植大鼠创面后的效果。方法构建PDGFB真核表达质粒,在脂质体介导下转染大鼠成纤维细胞。分别构建复合皮1(角质形成细胞 猪脱细胞真皮基质 PDGFB基因转染的成纤维细胞)和复合皮2(角质形成细胞 猪脱细胞真皮基质 未转染的成纤维细胞),移植于大鼠背部全层皮肤缺损创面,相应设为转染组、未转染组(各18只)。以不作皮肤移植的8只大鼠全层皮肤缺损创面为对照组。术后2周观察大鼠创面移植皮片存活情况。术后2、4、6周观察大鼠创面大体情况,计算创面收缩率,并取创面组织标本进行组织学观察。结果(1)术后2周,转染组大鼠中皮片完全存活者14只、部分存活者3只、未存活者1只;未转染组大鼠中皮片完全存活者10只、部分存活者4只、未存活者4只。(2)术后2周,对照组创面结痂。术后6周转染组移植皮片表面光滑,有弹性,抗磨擦性强,愈合效果优于其他两组。(3)术后2、4、6周,对照组大鼠创面收缩率均高于其他两组,转染组创面收缩率低于未转染组(P<0.05)。(4)术后2周转染组移植皮片内可见较多毛细血管分布;6周时表皮细胞分化达7～10层,纤维排列致密整齐,毛细血管分布均匀。结论用含PDGFB基因的人工复合皮移植修复创面,可明显提高创面愈合质量。     

Regression of advanced diabetic nephropathy by hepatocyte growth factor gene therapy in rats

Diabetic nephropathy is the main cause of end-stage renal disease requiring dialysis in developed countries. In this study, we demonstrated the therapeutic effect of hepatocyte growth factor (HGF) on advanced rather than early diabetic nephropathy using a rat model of streptozotocin-induced diabetes. Early diabetic nephropathy (16 weeks after induction of diabetes) was characterized by albuminuria, hyperfiltration, and glomerular hypertrophy, whereas advanced diabetic nephropathy showed prominent transforming growth factor (TGF)-beta1 upregulation, mesangial expansion, and glomerulosclerosis. An SP1017-formulated human HGF (hHGF) plasmid was administered by intramuscular injection combined with electroporation over a 30-day follow-up in rats with early and advanced diabetic nephropathy. hHGF gene therapy upregulated endogenous rat HGF in the diabetic kidney (rat HGF by RT-PCR was threefold higher than in diabetic rats without therapy). hHGF gene therapy did not improve functional or morphologic abnormalities in early diabetic nephropathy. hHGF gene therapy reduced albuminuria and induced strong regression of mesangial expansion and glomerulosclerosis in advanced diabetic nephropathy. These findings were associated with suppression of renal TGF-beta1 and mesangial connective tissue growth factor (CTGF) upregulation, inhibition of renal tissue inhibitor of metalloproteinase (TIMP)-1 expression, and reduction of renal interstitial myofibroblasts. In conclusion, our results suggest that hHGF gene therapy may be considered as an innovative therapeutic strategy to treat advanced diabetic nephropathy.     

PDGF-B基因表达在组织工程化皮肤移植后血管重建中的作用

目的构建含有血小板衍化生长因子B(PDGFB)基因的组织工程化皮肤,进行动物移植实验,研究PDGFB基因表达在真皮血管重建中的作用。方法构建PDGFB真核表达质粒,用脂质体LipofectAMINE介导转染成纤维细胞。分别构建3种不同类型的组织工程化皮肤角质形成细胞 脱细胞猪真皮基质(A组),角质形成细胞 脱细胞猪真皮基质 成纤维细胞(B组),角质形成细胞 脱细胞猪真皮基质 PDGFB基因转染的成纤维细胞(C组),分别移植于大鼠背部创面,观察术后2、4、6周真皮血管重建情况。结果术后2周C组真皮浅层内可见较多新生毛细血管长入,B组次之,A组毛细血管长入较少(P<005);术后4周各组真皮浅层内毛细血管数逐渐增加,但C组毛细血管数仍明显高于B、A两组(P<005);术后6周各组织工程化皮肤内毛细血管数差异无显著性意义。结论PDGFB基因在组织工程化皮肤移植后早期真皮血管重建中发挥了重要的作用,为移植后皮片成活提供了保障。     

Nerve growth factor accelerates wound healing in diabetic mice.

Patients with diabetic neuropathy have reduced numbers of cutaneous nerves, which may contribute to an increased incidence of nonhealing wounds. Nerve growth factor (NGF) has been reported to augment wound closure. We hypothesized that topical 2.5S NGF, a biologically active subunit of the NGF polymer, would accelerate wound repair, augment nerve regeneration, and increase inflammation in excisional wounds in diabetic mice. A full-thickness 6-mm punch biopsy wound was created on the dorsum of C57BL/6J-m+ Leprdb mice (db/db) and heterozygous (db/-) littermates and treated daily with normal saline or 2.5S NGF (1 microg/day or 10 microg/day) on post-injury days 0-6. Time to closure, wound epithelialization, and degree of inflammation were compared using a Student's t-test. Color subtractive-computer-assisted image analysis was used to quantify immunolocalized nerves in wounds. Non-overlapping (20x) digital images of the wound were analyzed for nerve profile counts, area density (number of protein gene product 9.5 positive profiles per unit dermal area) and area fraction (protein gene product 9.5 positive area per unit dermal area). Healing times in db/db mice decreased from 30 days in normal saline-treated mice to 26 days in mice treated with 1 microg/day NGF (p<0.05) and 24 days in mice treated with 10 microg/day NGF (p<0.02). A similar trend in db/- mice was not significant. NGF treatment augmented epithelialization in the db/db mice (p<0.05). Histological evaluation of inflammation in healed wounds showed no statistical difference between treatment groups. Total nerve number, area density, and area fraction were increased in NGF-treated wounds at 14, 21, and 35 days (p<0.05). The 2.5 NGF subunit may improve wound closure kinetics by promoting epithelialization and nerve regeneration. Further studies to determine the role of nerves in wound repair are warranted.