Accelerate Healing of Severe Burn Wounds by Mouse Bone Marrow Mesenchymal Stem Cell-Seeded Biodegradable Hydrogel Scaffold Synthesized from Arginine-Based Poly(ester amide) and Chitosan.

Severe burns are some of the most challenging problems in clinics and still lack ideal modalities. Mesenchymal stem cells (MSCs) incorporated with biomaterial coverage of burn wounds may offer a viable solution. In this report, we seeded MSCs to a biodegradable hybrid hydrogel, namely ACgel, that was synthesized from unsaturated arginine-based poly(ester amide) (UArg-PEA) and chitosan derivative. MSC adhered to ACgels. ACgels maintained a high viability of MSCs in culture for 6 days. MSC seeded to ACgels presented well in third-degree burn wounds of mice at 8 days postburn (dpb) after the necrotic full-thickness skin of burn wounds was debrided and filled and covered by MSC-carrying ACgels. MSC-seeded ACgels promoted the closure, reepithelialization, granulation tissue formation, and vascularization of the burn wounds. ACgels alone can also promote vascularization but less effectively compared with MSC-seeded ACgels. The actions of MSC-seeded ACgels or ACgels alone involve the induction of reparative, anti-inflammatory interleukin-10, and M2-like macrophages, as well as the reduction of inflammatory cytokine TNFα and M1-like macrophages at the late inflammatory phase of burn wound healing, which provided the mechanistic insights associated with inflammation and macrophages in burn wounds. For the studied regimens of these treatments, no toxicity was identified to MSCs or mice. Our results indicate that MSC-seeded ACgels have potential use as a novel adjuvant therapy for severe burns to complement commonly used skin grafting and, thus, minimize the downsides of grafting.

Surgical Denervation of Specific Cutaneous Nerves Impedes Excisional Wound Healing of Small Animal Ear Pinnae.

Damage to cutaneous nerves inhibits wound healing in patients. Results from animals on the nerve contributions to healing are various and sometimes contradictory. Here, we aim to clearly define the collective role of central, caudal, and rostral nerves in ear wound healing of mice, rats, and rabbits. These wounds heal with minimal contraction like wounds in humans. We resected central, caudal, and rostral nerves at the base of ear pinnae by microsurgery and created excisional full-thickness skin wounds in the pinnae neurologically downstream from the resections. Denervation in mice resulted in no closure for 14 days post-wounding (dpw) and led to only 17.2% closure at 21 dpw when the excisional wounds of non-denervated ear pinnae were completely closed. Compared to excisional wounds that were not denervated in sham surgery, wounds with denervation showed an increase of excisional wound areas for 5.0% by 7 dpw and a 43.7% reduction of wound closure at 12 dpw for rats. In rabbits, denervation attenuated wound closure for 14.2, 34.4, and 28.3% at 7, 14, and 18 dpw, respectively. Our histological analysis showed marked denervation impairment in pivotal healing processes, re-epithelialization and granulation tissue growth, suggesting denervation impairment in the regeneration of blood capillaries and/or connective tissue in wounds. These results reveal the critical contributions made by central, caudal, and rostral nerves in ear pinnae to minimal-contraction skin wound healing. Our study also provides small animal models of minimal-contraction wound healing of denervated ear skins that recapitulate human wound healing involving surgical or traumatic nerve damages.

Neuroprotectin/protectin D1: endogenous biosynthesis and actions on diabetic macrophages in promoting wound healing and innervation impaired by diabetes.

Dysfunction of macrophages (MΦs) in diabetic wounds impairs the healing. MΦs produce anti-inflammatory and pro-resolving neuroprotectin/protectin D1 (NPD1/PD1, 10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid); however, little is known about endogenous NPD1 biosynthesis by MΦs and the actions of NPD1 on diabetic MΦ functions in diabetic wound healing. We used an excisional skin wound model of diabetic mice, MΦ depletion, MΦs isolated from diabetic mice, and mass spectrometry-based targeted lipidomics to study the time course progression of NPD1 levels in wounds, the roles of MΦs in NPD1 biosynthesis, and NPD1 action on diabetic MΦ inflammatory activities. We also investigated the healing, innervation, chronic inflammation, and oxidative stress in diabetic wounds treated with NPD1 or NPD1-modulated MΦs from diabetic mice. Injury induced endogenous NPD1 biosynthesis in wounds, but diabetes impeded NPD1 formation. NPD1 was mainly produced by MΦs. NPD1 enhanced wound healing and innervation in diabetic mice and promoted MΦs functions that accelerated these processes. The underlying mechanisms for these actions of NPD1 or NPD1-modulated MΦs involved 1) attenuating MΦ inflammatory activities and chronic inflammation and oxidative stress after acute inflammation in diabetic wound, and 2) increasing MΦ production of IL10 and hepatocyte growth factor. Taken together, NPD1 appears to be a MΦs-produced factor that accelerates diabetic wound healing and promotes MΦ pro-healing functions in diabetic wounds. Decreased NPD1 production in diabetic wound is associated with impaired healing. This study identifies a new molecular target that might be useful in development of more effective therapeutics based on NPD1 and syngeneic diabetic MΦs for treatment of diabetic wounds.

Maresin-like lipid mediators are produced by leukocytes and platelets and rescue reparative function of diabetes-impaired macrophages.

Nonhealing diabetic wounds are associated with impaired macrophage (Mf) function. Leukocytes and platelets (PLT) play crucial roles in wound healing by poorly understood mechanisms. Here we report the identification and characterization of the maresin-like(L) mediators 14,22-dihydroxy-docosa-4Z,7Z,10Z,12E,16Z,19Z-hexaenoic acids, 14S,22-diHDHA (maresin-L1), and 14R,22-diHDHA (maresin-L2) that are produced by leukocytes and PLT and involved in wound healing. We show that 12-lipoxygenase-initiated 14S-hydroxylation or cytochrome P450 catalyzed 14R-hydroxylation and P450-initiated ω(22)-hydroxylation are required for maresin-L biosynthesis. Maresin-L treatment restores reparative functions of diabetic Mfs, suggesting that maresin-Ls act as autocrine/paracrine factors responsible for, at least in part, the reparative functions of leukocytes and PLT in wounds. Additionally, maresin-L ameliorates Mf inflammatory activation and has the potential to suppress the chronic inflammation in diabetic wounds caused by activation of Mfs. These findings provide initial insights into maresin-L biosynthesis and mechanism of action and potentially offer a therapeutic option for better treatment of diabetic wounds.

Immunohistological localization of endogenous unlabeled stem cells in wounded skin.

Various types of endogenous stem cells (SCs) participate in wound healing in the skin at different anatomical locations. SCs need to be identified through multiple markers, and this is usually performed using flow cytometry. However, immunohistological identification of endogenous stem cells in the skin at different anatomical locations by co-staining multiple SC markers has been seldom explored. We examined the immunohistological localization of four major types of SCs in wounded skin by co-staining for their multiple markers. Hematopoietic SCs were co-stained for Sca1 and CD45; mesenchymal SCs for Sca1, CD29, and CD106; adipose SCs for CD34, CD90, and CD105; and endothelial progenitor cells and their differentiated counterparts were co-stained for CD34, Tie2, and von Willebrand factor. We found Sca1(+)CD45(+) SCs in the epidermis, dermis and hypodermis of wounded skin. Sca1(+)CD29(+) and Sca1(+)CD106(+) mesenchymal SCs, CD34(+)CD105(+), CD34(+)CD90(+), and CD90(+)CD105(+) adipose SCs, as well as CD34(+)Tie2(+) endothelial progenitor cells were also located in the epidermis, dermis, and hypodermis. This study demonstrates the feasibility of using immunohistological staining to determine the location of SCs in wounded skin and the intracellular distribution of their molecular markers.

The unfolded protein response is activated in connexin 50 mutant mouse lenses.

The unfolded protein response is a set of cell signaling pathways recently recognized to be activated in the lens during both normal development and endoplasmic reticulum stress induced by either unfolded proteins or oxidative damage. While mutations in the gene for connexin 50 are known to cause autosomal dominant cataracts, it has not been previously reported whether mutant connexins can activate the unfolded protein response in the lens. Mice homozygous for the S50P or G22R mutation of connexin 50 have reduced amounts of connexin 50 protein at the cell membrane, with some intracellular staining consistent with retention in the endoplasmic reticulum. Connexin 50 mutants have elevated levels of BiP expression in both lens epithelial and fiber cells from E15.5 with the most robust elevation detected in newborns. While this elevation decreases in magnitude postnatally, BiP expression is still abnormally high in adults, particularly in the perinuclear endoplasmic reticulum of cell nuclei that are inappropriately retained in adult homozygous mutant lenses. Xbp1 splicing was elevated in lenses from both connexin mutants studied, while Atf4 and Atf6 levels were not majorly affected. Overall, these data suggest that UPR may be a contributing factor to the phenotype of connexin 50 mutant lenses even though the relatively modest extent of the response suggests that it is unlikely to be a major driver of the pathology.

Matrix metalloproteinase-2 and -9 activities in the human lens epithelial cells and serum of steroid induced posterior subcapsular cataracts.

PURPOSE: To evaluate the level of matrix metalloproteinase (MMP)-2 and MMP-9 activities in patients with steroid induced posterior subcapsular cataract (PSC). METHODS: This prospective, observational study comprised of 156 patients having either steroid induced PSC (n=50) or non-steroidal PSC (n=106) were performed to evaluate the level of MMP-2 and MMP-9 activities in the lens epithelial cells (LECs) and the serum. Anterior lens capsules harboring LECs were obtained during phacoemulsification and peripheral blood was collected from patients before administration of anesthesia. Serum was separated by centrifugation at 10,000× g for 15 min at 4 °C. The LECs and serum samples were processed to analyze MMP-2 and MMP-9 activities using succinylated gelatin assay. Quantitative real time-PCR (qRT-PCR) was performed to determine the mRNA levels of MMP-2 and MMP-9 in LECs. The mRNA levels were expressed as a ratio, using the delta-delta method for comparing the relative expression results between cases with steroid induced PSC and cases with non-steroidal PSC. MMP-2 and MMP-9 levels were also compared in the two groups using immunolocalization. RESULTS: The level of MMP-2 and MMP-9 activity was found to be high in LECs and serum of cases with steroid induced PSC. Further in all steroid induced cases, a 1.4 fold increase was observed in MMP-2 activity in LECs and a 1.4 fold increase in MMP-9 activity in the serum. Both qRT-PCR and immunolocalization showed increased expression of MMP-2 and MMP-9 activity. CONCLUSIONS: MMP-2 and MMP-9 activity in both LECs and serum was significantly higher in cases with steroid induced PSC. The possible use of MMP-9 as a non-invasive biomarker in ascertaining the presence of steroid induced PSC should be evaluated using a larger sample size.

Matrix metalloproteinase-9 activity in human lens epithelial cells of cortical, posterior subcapsular, and nuclear cataracts.

PURPOSE: To evaluate the level of matrix metalloproteinase-9 (MMP-9) activity in lens epithelial cells (LECs) derived from different types of cataract in patients having phacoemulsification. SETTING: Iladevi Cataract & IOL Research Centre, Memnagar, Ahmedabad, India. METHODS: This observational study of 275 patients having phacoemulsification was performed to evaluate the level of MMP-9 activity in LECs. All anterior lens capsules harboring the LECs and derived from the surgical curvilinear capsulorhexis were obtained during phacoemulsification. The anterior lens capsule samples were processed to analyze MMP-9 activity using a succinylated gelatin assay. The samples were grouped based on age and on pure cataract type. RESULTS: The level of MMP-9 activity in LECs was highest in eyes with cortical cataract. A significant difference in the level of MMP-9 activity was found in different types of cataract (P<.001). The highest level of MMP-9 activity was in patients older than 60 years. The MMP-9 activity increased gradually with age irrespective of cataract type (P<.001). CONCLUSIONS: A significant difference was observed in the level of MMP-9 activity between different types of cataract. The activity of MMP-9 was highest in eyes with cortical cataract. The level of MMP-9 activity increased with age in the LECs of patients with age-related cataract.