A preliminary evaluation of the functional significance of alpha-1-acid glycoprotein glycosylation on wound healing

The laying down of collagen and fibrous tissue is a key process in wound healing, however excessive collagen (and glycoprotein) deposition causes hypertrophic and keloid scars, eg after burns. Collagen synthesis is increased in these scars compared with normal healing, as is collagenase activity, which controls the degradation pathway of collagen. The processes of wound healing are inextricably linked to those of the acute-phase response (APR): alpha-1-acid glycoprotein (AGP), a plasma glycoprotein that undergoes both an increase in concentration and an alteration in its glycosylation pattern during the APR. This study determined that AGP isolated from the plasma of burns patients was of an increased concentration and altered glycosylation pattern compared with normal plasma and was capable of directly interacting with type I collagen. It also had a profound effect on both collagen fibril formation and collagenase activity, to a degree dependent upon the percentage body surface area burned. Additionally, the results obtained provided the basis for predicting the formation of hypertrophic scars.     

Low-power laser irradiation improves histomorphometrical parameters and bone matrix organization during tibia wound healing in rats

The influence of daily energy doses of 0.03, 0.3 and 0.9 J of He-Ne laser irradiation on the repair of surgically produced tibia damage was investigated in Wistar rats. Laser treatment was initiated 24 h after the trauma and continued daily for 7 or 14 days in two groups of nine rats (n=3 per laser dose and period). Two control groups (n=9 each) with injured tibiae were used. The course of healing was monitored using morphometrical analysis of the trabecular area. The organization of collagen fibers in the bone matrix and the histology of the tissue were evaluated using Picrosirius-polarization method and Masson's trichrome. After 7 days, there was a significant increase in the area of neoformed trabeculae in tibiae irradiated with 0.3 and 0.9 J compared to the controls. At a daily dose of 0.9 J (15 min of irradiation per day) the 7-day group showed a significant increase in trabecular bone growth compared to the 14-day group. However, the laser irradiation at the daily dose of 0.3 J produced no significant decrease in the trabecular area of the 14-day group compared to the 7-day group, but there was significant increase in the trabecular area of the 15-day controls compared to the 8-day controls. Irradiation increased the number of hypertrophic osteoclasts compared to non-irradiated injured tibiae (controls) on days 8 and 15. The Picrosirius-polarization method revealed bands of parallel collagen fibers (parallel-fibered bone) at the repair site of 14-day-irradiated tibiae, regardless of the dose. This organization improved when compared to 7-day-irradiated tibiae and control tibiae. These results show that low-level laser therapy stimulated the growth of the trabecular area and the concomitant invasion of osteoclasts during the first week, and hastened the organization of matrix collagen (parallel alignment of the fibers) in a second phase not seen in control, non-irradiated tibiae at the same period. The active osteoclasts that invaded the regenerating site were probably responsible for the decrease in trabecular area by the fourteenth day of irradiation.     

Elemental composition of hypertrophic scar and normal skin tissue using proton induced X-ray emission

Hypertrophic scars are a particular type of scar that can form after any type of dermal trauma. They are unsightly, red and elevated above normal skin level. At present no-one knows why these scars form and what form the treatment should take. Full thickness hypertrophic skin tissue as well as full thickness normal skin samples, obtained form the Restoration of Appearance and Function Trust (RAFT), Institute of Plastic Surgery, Mount Vernon Hospital, were analysed, using simultaneously both PIXE and RBS, with a 2 MeV proton beam. The epidemis was compared to the dermis on both normal and scarred tissue, and each was compared to the other, to see if there were any variations in elemental composition. In all the samples C, N and O detected by RBS and P, S, Cl, K, Ca, Fe detected by PIXE were found, In the majority of samples Zn and Cd were found, and in a few samples Sn was determined. Significant differences in concentrations, for the elements P, S, K and Cd, between the epidermis and dermis in both hypertrophic scarred and normal skin tissue were found. A difference was also detected between elemental concentrations in normal and scarred skin for the elements Ca, P, S, Fe and Cd.     

Collagen Peptides Derived from Sipunculus nudus Accelerate Wound Healing

Marine collagen peptides have high potential in promoting skin wound healing. This study aimed to investigate wound healing activity of collagen peptides derived from Sipunculus nudus (SNCP). The effects of SNCP on promoting healing were studied through a whole cortex wound model in mice. Results showed that SNCP consisted of peptides with a molecular weight less than 5 kDa accounted for 81.95%, rich in Gly and Arg. SNCP possessed outstanding capacity to induce human umbilical vein endothelial cells (HUVEC), human immortalized keratinocytes (HaCaT) and human skin fibroblasts (HSF) cells proliferation and migration in vitro. In vivo, SNCP could markedly improve the healing rate and shorten the scab removal time, possessing a scar-free healing effect. Compared with the negative control group, the expression level of tumor necrosis factor-α, interleukin-1β and transforming growth factor-β1 (TGF-β1) in the SNCP group was significantly down-regulated at 7 days post-wounding (p < 0.01). Moreover, the mRNA level of mothers against decapentaplegic homolog 7 (Smad7) in SNCP group was up-regulated (p < 0.01); in contrast, type II TGF-β receptors, collagen I and α-smooth muscle actin were significantly down-regulated at 28 days (p < 0.01). These results indicate that SNCP possessed excellent activity of accelerating wound healing and inhibiting scar formation, and its mechanism was closely related to reducing inflammation, improving collagen deposition and recombination and blockade of the TGF-β/Smads signal pathway. Therefore, SNCP may have promising clinical applications in skin wound repair and scar inhibition.     

The effect of 880 nm low level laser energy on human fibroblast cell numbers: a possible role in hypertrophic wound healing

Low level lasers (LLLs) have been shown to induce therapeutic effects in wound healing. However, there have been few LLL studies on burn wounds which may become unsightly, hypertrophic and impair function. Inhibitory effects on the healing of fibrotic wounds, prone to hypertrophy may be expected to reasonably reduce the problems accompanying hypertrophic scarring. The effects of LLL wavelengths and treatment parameters on wound healing cells in vitro often demonstrate meaningful results and without concurrent ethical difficulties of clinical trials. This experiment investigated the effect of an 880 nm, 16 mW GaAlAs diode at 2.4 and 4 J/cm(2) on cell numbers of two human fibroblast cell lines, derived from hypertrophic scar (HF) and normal dermal explants (NF), respectively. After irradiation by 880 nm LLL, cell numbers were measured utilising methylene blue bioassay and read by the spectrophotometer in the same microculture plates. HF and NF exhibited decreased cell numbers as compared to sham-irradiated controls. HF cell number, after 2.4 J/cm(2), was significantly lower on day 5 (P<0.05). The NF cell numbers were significantly lower on day 4 and/or day 5 (P<0.05). The results have implications on hypertrophic wound healing and further studies are required.     

Evaluation of Wound-Healing and Antioxidant Effects of Marantodes pumilum (Blume) Kuntze in an Excision Wound Model

Marantodes pumilum (MP) is a great source of herbal medicine used traditionally by both men and women for various purposes. MP may have potential wound-healing effects due to its diverse biological properties. An extensive study was conducted in a normal male rat model for determining the effects of MP var. pumila (MPvp) and var. alata (MPva) on the wound healing process. Here, 126 male Sprague-Dawley rats were divided randomly into seven groups as follows: sham-operated (SH), vehicle dressing (VD), flavine dressing (FD), MPvp leaves (PL), MPvp roots (PR), MPva leaves (AL), and MPva roots (AR). The parameters studied were the percentage of wound contraction, histomorphology study by hematoxylin and eosin (H&E), Masson–Goldner trichrome (MGT), and immunohistochemistry (IHC) staining. In addition, the levels of enzymatic antioxidants and malondialdehyde were also measured in the wound tissue homogenates. Wounds treated with extracts (PL, PR, AL, and AR) showed significantly faster healing (p < 0.05) compared to untreated and control groups (SH, VD, and FD). Histological analysis among MP-treated groups revealed better re-epithelialization, higher collagen deposition, enhanced fibronectin content and fibroblast cells, and higher fiber transformation from collagen-III to collagen-I, accompanied with a significant surge in enzymatic antioxidant activities and a decline in lipid peroxidation. MP has antioxidant effects that may enhance wound healing in the rat model.     

Differential scanning calorimetric examination of pathologic scar tissues of human skin

Keloid and hypertrophic scarring is a dermal fibroproliferative disorder characterized by increased fibroblast proliferation and excessive production of collagen. Excess scar formation occurs after dermal injury as a result of abnormal wound healing. Keloid formation has been ascribed to altered growth factor regulation, aberrant collagen turnover, genetics, immune dysfunction, sebum reaction, and altered mechanics. No single hypothesis adequately explains keloid formation. The thermal denaturations of pathologic human skin scar tissues were monitored by a SETARAM Micro DSC-II calorimeter. All the experiments were performed between 0 and 100 °C. The heating rate was 0.3 K min^?1. DSC scans clearly demonstrated significant differences between the different types and conditions of samples (intact skin: T _m = 54.8 °C and ΔH _cal = 4.5 J g^?1; normal scar: T _m = 53.8 °C and ΔH _cal = 4.2 J g^?1; hypertrophic scar: T _m = 54.2 °C and ΔH _cal = 2.4 J g^?1; keloid: T _m = 52.9 °C and ΔH _cal = 8.3 J g^?1). The heat capacity change between native and denatured states of samples increased with the degree of structural alterations indicating significant water loosing. These observations could be explained with the structural alterations caused by the biochemical processes. With our investigations, we could demonstrate that DSC is a useful and well-applicable method for the investigation of collagen tissue of the human keloid and hypertrophic scar tissues. Our results may be of clinical relevance in the future, i.e., in the diagnosis of the two different pathologic scar formations, or in the choice of the optimal therapy of the disease.     

Influence of laser photobiomodulation upon connective tissue remodeling during wound healing

The modulation of collagen fibers during experimental skin wound healing was studied in 112 Wistar rats submitted to laser photobiomodulation treatment. A standardized 8mm-diameter wound was made on the dorsal skin of all animals. In half of them, 0.2ml of a silica suspension was injected along the border of the wound in order to enhance collagen deposition and facilitate observation. The others received saline as vehicle. The treatment was carried out by means of laser rays from an aluminum-gallium arsenide diode semiconductor with 9mW applied every other day (total dose=4J/cm(2)) on the borders of the wound. Tissue sections obtained from four experimental groups representing sham-irradiated animals, laser, silica and the association of both, were studied after 3, 7, 10, 15, 20, 30 and 60 days from the laser application. The wounded skin area was surgically removed and submitted to histological, immunohistochemical, ultrastructural, and immunofluorescent studies. Besides the degree and arrangement of collagen fibers and of their isotypes, the degree of edema, the presence of several cell types especially pericytes and myofibroblasts, were described and measured. The observation of Sirius-red stained slides under polarized microscopy revealed to be of great help during the morphological analysis of the collagen tissue dynamic changes. It was demonstrated that laser application was responsible for edema regression and a diminution in the number of inflammatory cells (p<0.05). An evident increase in the number of actin-positive cells was observed in the laser-treated wounds. Collagen deposition was less than expected in silica-treated wounds, and laser treatment contributed to its better differentiation and modulation in all irradiated groups. Thus, laser photobiomodulation was able to induce several modifications during the cutaneous healing process, especially in favoring newly-formed collagen fibers to be better organized and compactedly disposed.     

Elemental composition of skin tissue by PIXE and INA analyses

Hypertrophic scars are a particular type of scar that can form after any type of dermal injury. They are unsightly, red, elevated above nomal skin level, itchy and painful. At present no-one knows why these scars form. Due to the differences between scar tissue and normal skin it was decided to analyse skin tissue which at a later date would be compared to scar tissue. Split skin graft (epidemis) samples as well as full thickness skin (epidemis to demis) tissue were analysed using PIXE and RBS with a 2 MeV proton beam. The epidemis was compared to the demis to see if there were any variations between the two. In all the samples P, S, Cl, K, Ca, Fe, and Cd were detected and in a majority of the samples Zn and Ba were found, using PIXE. It was found that, between the epidemis and demis, there was an significant increase in P in the epidermis in both full thickness and split skin graft samples. Complementary elemental analysis was also performed using neutron activation, and Br, Na and Zn were detected.     

Study on the Influence of Nitrogen Plasma on Dyeing Properties of Rex Rabbit Fibers

The surface of rex rabbit fibers is hydrophobic in nature because of the presence of the hard cuticle on its surface, and this hydrophobicity may give rise to many problems in the dyeing and finishing processes. In order to improve its dyeability and decrease dye pollution in sewage discharge, nitrogen plasma was used to modify rabbit fibers and after that the modified fibers were dyed with the anionic dyestuffs (C.I. number 16185). The effects of nitrogen plasma on the dyeing properties and the dyeing behavior for the rex rabbit fibers were studied, the related parameters including the treatment time and discharge power were optimized. Surface morphology and roughness of rex rabbit fibers were characterized by scanning electron microscope and atomic force microscopy. XPS and FTIR-ATR were further performed to determine the surface chemical compositions of rex rabbit fibers. The physical properties of rex rabbit fibers were characterized by tensile strength tests. The results show that nitrogen plasma treatment can remove surface scales on the rex rabbit fibers and introduce more active groups such as hydroxyl (–OH), carbonyl (–C=O), and amino (–NH₂) on the surface of the fibers, which makes rex rabbit fibers have better dyeability, and effectively improves dyeing rate and dye fixation rate.     

Synergistic Effects of D‐Glucosamine and Collagen Peptides on Healing Experimental Cartilage Injury

We investigated the synergistic wound‐healing effects of D ‐glucosamine (GlcN) and collagen peptide ( : 10 000 Daltons soluble collagen (S‐Co)) on injured cartilage. A injured cartilage model in rabbit was surgically created. Under general anesthesia, three holes at articular cartilage of the medial trochlear (one hole) and trochlear sulcus (two holes) of a distal femur were created using a high‐speed orthopedic drill. After surgery, rabbits were orally administered with collagen peptide (S‐Co group), GlcN (G group), or both (CoG group) for 2 or 3 weeks. In addition, glycine (Gly group) was administered as a control for the S‐Co group in three rabbits. Control rabbits were provided with water ad lib. After 2 weeks, the holes were filled by fibroblasts and chondroblasts in the treatment group, whereas in the control group, the holes were not filled completely unlike for the treatment group. Even three weeks later, only a small change was seen in the control group. In the group receiving collagen and GlcN, the holes were completely filled by proliferating chondroblasts, remodeling of bony trabeculae was also observed. Image analysis of Safranin O‐ and Alcian blue‐stained specimens showed increased production of proteoglycans and glycosaminoglycans, respectively, not only in the injured site, but also in the normal cartilage in the treated groups. The most enhanced healing of injury and increase in Safranin O and Alcian blue positive materials was observed in the CoG group. As a result, simultaneous administration of collagen and GlcN gave a fairly enhanced healing on restoration of cartilage injuries.

Artificial cartilage injury at the stifle joint of a rabbit. a: proximal hole in trochlear sulcus; b: distal hole in trochlear sulcus; c: medial hole in medial trochlear ridge.     

Polylysine coated bacterial cellulose nanofibers as novel templates for bone-like apatite deposition

The study on bacterial cellulose (BC) nanofibers used as templates for hydroxylapatite (HAp) deposition has been investigated by our group and many other researchers. However, BC is only microscopically similar to natural collagen but not molecular structure. If protein could be introduced to the surfaces of BC nanofibers, the BC nanofibers could mimic the natural collagen fibers in terms of both shape and molecular structure. In this work, our latest results concerning the preparation of polylysine (PLL) coated BC nanofibers are reported. It is found that the ε-polylysine (PLL), a natural coming peptide, was introduced to the surfaces of BC nanofibers via crosslinking method by using procyanidins as crosslinker. The bioactivity of PLL coated BC nanofibers was demonstrated by the bone-like HAp deposition throughout the scaffold in a simulated body fluid (SBF). To initiate mineralization the PLL coated BC nanofibers were immersed in 1.5 times simulated body fluids (1.5 SBF) at 37 °C for 7 days. The deposited minerals on the nanofiber surfaces were characterized using scanning electron microscopy (SEM), X-ray diffraction (XRD), and fourier transformed infrared spectroscopy (FTIR). These PLL coated BC nanofibers were proved to act as nano templates to induce the formation of nano-sized platelet-like, calcium-deficient, B-type carbonated HAp of which the features was closed to those of biological apatite.     

The effects of low dose leukotriene receptor antagonist therapy on airway remodeling and cysteinyl leukotriene expression in a mouse asthma model

Airway structural changes that occur in patients with asthma in response to persistent inflammation are termed airway remodeling. The cysteinyl leukotrienes (LTC(4), D(4) and E(4)) are known to play important roles in the pathobiology of asthma. To evaluate the effect of low dose montelukast (MK) on the development of airway remodeling using a chronic murine model of allergic airway inflammation with subepithelial fibrosis, BALB/c mice, after intraperitoneal ovalbumin (OVA) sensitization on days 0 and 14, received intranasal OVA periodically on days 14-75. MK treated mice received montelukast sodium intraperitoneally on days 26-75. The OVA sensitized/challenged mice developed an extensive eosinophil cell inflammatory response, goblet cell hyperplasia, mucus occlusion, and smooth muscle hypertrophy of the airways. In addition, in OVA sensitized/challenged mice, dense collagen deposition/fibrosis was seen throughout the lung interstitium surrounding the airways, blood vessels, and alveolar septae. The cysteinyl leukotriene 1 (CysLT1) receptor antagonist, MK significantly reduced the airway eosinophil infiltration, goblet cell hyperplasia, mucus occlusion, and lung fibrosis except airway smooth muscle hypertrophy in the OVA sensitized/challenged mice. The OVA sensitized/challenged mice had significantly increased epithelial desquamation compared with control mice. MK markedly reduced epithelial desquamation of airways in OVA/MK treated animals compared with OVA sensitized/challenged mice. MK treatment did not affect the levels of CysLT in lung tissue. Our results show that the important role of cysteinyl leukotrienes in the pathogenesis of asthma. Lower dose of CysLT1 receptor antagonism has a significant anti-inflammatory effect on allergen-induced lung inflammation and fibrosis but not airway smooth muscle hypertrophy in an animal model of asthma.     

Effects of adenovirus-mediated bFGF, IL-1Ra and IGF-1 gene transfer on human osteoarthritic chondrocytes and osteoarthritis in rabbits

The study investigated the effects of adenovirus-mediated gene transfection of basic fibroblast growth factor (bFGF), bFGF combined with interleukin-1 receptor antagonist protein (IL-Ra) and/or insulin-like growth factor-1 (IGF-1) both in human osteoarthritis (OA) chondrocytes and rabbits OA model. Human OA chondrocytes were delivered by adenovirus-mediated bFGF, IL-Ra and IGF-1 vectors, respectively. Chondrocyte proliferation, glycosaminoglycan (GAG) content, expression of type II collagen, ADAMTS-5, MMP-13, MMP-3 and TIMP-1 were determined. Rabbit OA model was induced by anterior cruciate ligament transaction (ACLT) in knees. Adenoviral vectors encoding human bFGF, IL-Ra and IGF-1 were injected intraarticularly into the knee joints after ACLT. The effects of adenovirus- mediated gene transfection on rabbit OA were evaluated. In vitro, the transfected genes were expressed in cell supernatant of human OA chondrocytes. AdbFGF group significantly promoted chondrocyte proliferation, and increased GAG and type II collagen synthesis than in the OA group. As two or three genes were transfected in different combinations, there was significant enhancement on the GAG content, type II collagen synthesis, and TIMP-1 levels, while ADAMTS-5, MMP-13, and MMP-3 levels were reduced. In vivo, the transfected genes were expressed in synovial fluid of rabbits. Intraarticular delivery of bFGF enhanced the expression of type II collagen in cartilage and decreased cartilage Mankin score compared with the OA control group (P = 0.047; P < 0.01, respectively). Multiple-gene transfection in different combinations showed better results than bFGF transfection alone. This study suggests that bFGF gene transfection is effective in treating experimental OA. Multiple gene transfection has better biologic effects on OA.     

Effect of a scaffold fabricated thermally from acetylated PLGA on the formation of engineered cartilage

A MHDS has been employed to fabricate 3D scaffolds from PLGA with acetyl endgroups to achieve in vivo regeneration of cartilage tissue. The fabricated acetylated-PLGA scaffold showed open pores and interconnected structures. Rabbit chondrocytes were seeded on the PLGA scaffolds and transplanted immediately into subcutaneous sites of athymic mice. Chondrocytes transplantation with untreated PLGA scaffolds served as a control. Histological analysis of the implants at 4 weeks with H&E staining and alcian blue staining revealed higher extracellular matrix and GAG expression at the neocartilage in the PLGA-6Ac scaffolds than that of the PLGA-6OH scaffold group. This endgroup-modified scaffold may be useful for successful cartilage tissue engineering in orthopedic applications.     

Grain boundary scars on spherical crystals

We present an experimental system suitable for producing spherical crystals and for observing the distribution of lattice defects (disclinations and dislocations) on a significant fraction (50%) of the sphere. The introduction of fluorescently labeled particles enables us to determine the location and orientation of grain boundary scars. We find that the total number of scars and the number of excess dislocations per scar agree with theoretical predictions and that the geometrical centers of the scars are roughly positioned at the vertices of an icosahedron.     

Osmolyte Type and the Osmolarity Level Affect Chondrogenesis of Mesenchymal Stem Cells

The inductive effects of increased osmolarity on chondrogenesis are well approved. However, the effects of the osmolyte agent invoked to induce hyperosmolarity are largely neglected. Herein, we scrutinized how hyperosmotic conditions acquired by addition of different osmolytes would impact chondrogenesis. We briefly assessed whether such conditions would differentially affect hypertrophy and angiogenesis during MSC chondrogenesis. Chondrogenic and hypertrophic marker expression along with VEGF secretion during adipose-derived (AD)-MSC chondrogenesis under three osmolarity levels (350, 450, and 550 mOsm) using three different osmolytes (NaCl, sorbitol, and PEG) were assessed. MTT assay, qRT-PCR, immunocytochemistry, Alcian Blue staining, ELISA, and ALP assays proved osmolyte-type dependent effects of hyperosmolarity on chondrogenesis, hypertrophy, and angiogenesis. At same osmolarity level, PEG had least cytotoxic/cytostatic effect and most prohibitive effects on angiogenesis. As expected, all hyperosmolar conditions led to enhanced chondrogenesis with slightly varying degrees. PEG and sorbitol had higher chondro-promotive and hypertrophy-suppressive effects compared to NaCl, while NaCl had exacerbated hypertrophy. We observed that TonEBP was involved in osmoadaptation of all treatments in varying degrees. Of importance, we highlighted differential effects of hyperosmolarity obtained by different osmolytes on the efficacy of chondrogenesis and more remarkably on the induction/suppression of cartilage pathologic markers. Our study underlies the need for a more vigilant exploitation of physicobiochemical inducers in order to maximize chondrogenesis while restraining unwanted hypertrophy and angiogenesis.     

The Application of the Starfish Hatching Enzyme for the Improvement of Scar and Keloid Based on the Fibroblast-Populated Collagen Lattice

Various bioactivities of the starfish hatching enzyme (HE) including collagen gel contraction, MMPs activity, hydroxyproline release, and gene regulation based on the fibroblast-populated collagen lattice (FPCL) in three-dimensional medium were investigated for the improvement of scar and keloid. The starfish HE significantly inhibited the collagen gel contraction over 2 days of culture. MMP-2 and MMP-9 activities were also identified by gelatin zymography and RT-PCR products with both HE and collagenase treatments, which resulted in the high amount of hydroxyproline release. The HE treatment on the FPCL significantly inhibited the fibroblast proliferation at 3 days of culture. The LPS-induced NO level and iNOS mRNA expression at low concentrations of HE presented a certain ability to inflammatory response. The COX-2 mRNA from the FPCL indicated no significant inflammation-mediated activity at 5 μg/mL of HE, whereas the cytokines of TNF-α and IL-1β were significantly higher than those of the control. Hence, the starfish hatching enzyme can regulate the fibroblast-populated collagen gel conditions by the contraction, MMP production, inflammatory gene expression, etc. Therefore, the starfish HE could be a potential cosmeceutical to heal the scar and keloid tissue.