Freeze-dried Skin Research Articles

Biophysical and permeability characterization of lyophilized (freeze-dried) human cadaver skin

The in vitro permeation testing (IVPT) of topical products is performed across the human cadaver skin, which is stored frozen for a prolonged duration. The cryo-preservation technique is not economical and is a cumbersome process. Moreover, prolonged skin preservation in a frozen state and frequent freeze-thawing are known to affect the integrity of the skin barrier. Therefore, lyophilization was explored as an alternative to protect the skin tissue from microbial contamination and degeneration. Notably, the project's objective was to investigate the impact of the freeze-drying process on the skin's barrier properties. The morphometrics of the lyophilized skin were measured. Histological studies did not reveal any notable changes in the organization and intactness of the layers due to the freeze-drying process. The biophysical attributes of the skin, such as transepidermal water evaporation rate and transepidermal electrical resistivity (TEER), were not significantly different between the control skin (not subjected to the freeze-drying process) and the freeze-dried skin (FDS). The permeability of caffeine, a hydrophilic model permeant, and nicotine, a lipophilic model permeant, were consistent across the control and the FDS. It is evident from the studies that the lyophilization process did not significantly impact the barrier properties and permeability of the skin.

Development of a Freeze-Dried Skin Care Product Composed of Hyaluronic Acid and Poly(γ-Glutamic Acid) Containing Bioactive Components for Application after Chemical Peels

Eight types of spongy sheet were prepared by freeze-drying aqueous solutions of hyaluronic acid (HA) and poly(γ-glutamic acid) (PGA) with or without bioactive components including vitamin C derivative (VC), glucosylceramide (GC), and epidermal growth factor (EGF). Spongy sheets were categorized into the following groups: Group I (HA/PGA), Group II (HA/PGA + VC), Group III (HA/PGA + GC), Group IV (HA/PGA + VC, GC), Group V (HA/PGA + EGF), Group VI (HA/PGA + VC, EGF), Group VII (HA/PGA + GC, EGF), and Group VIII (HA/PGA + VC, GC, EGF). In the first experiment, we examined fibroblast proliferation in conditioned medium that had been prepared by immersing each spongy sheet in a conventional culture medium. EGF-incorporating spongy sheets (Groups V-VIII) enhanced fibroblast proliferation more than EGF-free spongy sheets (Groups I-IV). In the second experiment, cytokine production by fibroblasts was evaluated using a wound surface model. This involved elevation of fibroblasts-incorporating collagen gel sheets to the air-liquid interface, on which a spongy sheet (Groups I, IV, V and VIII) was placed and cultured for 1 week. EGF-incorporating spongy sheets (Groups V and VIII) enhanced the production of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) by fibroblasts more than EGF-free spongy sheets (Groups I and IV). The effect of these four types of spongy sheet on wounds was investigated in animal experiments. Chemical peel was performed by contacting 50% trichloroacetic acid (TCA) on the dorsal region of mice, after which a spongy sheet was placed, and the wound condition was then observed in a two-week period. Angiogenesis was facilitated to a greater degree in Group VIII compared with Groups I, IV and V. This finding indicates that Group VIII spongy sheet is a promising aid for skin recovery after chemical peel.

English

  The purpose of this study was to explore the physicochemical and antioxidant characteristics of  Baccaurea angulata fruit juice extract. Freeze-dried whole fruit (FDWF), freeze-dried berry (FDB), and  freeze-dried skin (FDS) of B. angulata were analyzed for total phenolic, total flavonoid, total  anthocyanin, and antioxidant activities. FDS recorded the highest moisture and ash content, protein,  total fat, and water activity, compared to FDWF and FDB. FDS also contained the highest total phenolic,  total flavonoid, and total anthocyanin, while FDWF recorded the highest in scavenging xanthine oxidase  (35.9%) and ferric reducing activity (44.9 µM TE/g). FDS, however, showed the highest DPPH (102.66 mg  AA/100 g) and TEAC (847.46 mg TE/100 g) values. DPPH, TEAC and FRAP were strongly correlated with  total phenol (r = 0.979; 0.948; 0.997) and total flavonoid (r = 0.987; 0.960; 0.992). Total anthocyanin had  no correlation with DPPH and TEAC, but moderately with FRAP (r = 0.734). Physicochemical and  antioxidant characteristics of B. angulata may indicate that this fruit may impart health benefits when  consumed and can be suggested as a good source for nutraceutical beverages.     Key words: Antioxidant properties, Baccaurea angulata, freeze-dried fruit, nutritional composition,  physicochemical. 

What is new in burn care?

A burn injury is not limited to the skin but is associated with the dysfunction of almost every cell in the body and various organs leading to alterations, not only in cellular functions but also in cellular signaling pathways. The current thought that alterations and new treatments of these pathways may restore cellular function and productivity, which in turn may lead to an improved energy metabolism, cell survival, morbidity, and mortality is perhaps the most cutting-edge research in burn care. Hypermetabolism is associated with cell deaths (apoptosis) in skeletal muscles, fat, liver, kidney, lung, heart and gut. These detrimental cellular events are also associated with dysfunction of the cellular organelles such as the mitochondria or the endoplasmic reticulum, thus making burn a truly systemic disease. The areas of advancement in burn care that have improved results of both acute injuries as well as long-term outcome include acute care, burn wound treatment, control of the hypermetabolic responses, and control of life-threatening infections. Both the mortality as well as the length of hospital stay of burned patients have been greatly reduced because of improvement in acute care, such as resuscitation and management of inhalation injuries, early excision and grafting, the control of infections and improving the immune system, improvements in provision of metabolic and nutritional requirements, evolution of effective skin banks, infection control, and alternative wound-closure materials and strategies. Gene therapy is another emerging technique to alter dermal and epidermal regeneration and to improve wound healing. Non-viral constructs have been shown to effectively change the cells into a productive and active cell that releases growth factors and other signaling factors to improve dermal and epidermal regeneration. Liposomal gene constructs have been shown to transfect dermal stem cells increasing the therapeutic possibilities of this futuristic technology! Skin substitutes are creating a revolution and it is now possible to construct an artificial skin, which is called cultured skin substitute (CSS). CSS is the patients own skin (epithelium) in combination with a dermal substitute (Integra) that represents a full-skin substitute including both dermis as well as epidermis. The use of bilayer artificial skin has improved the survival and cosmetic results of early eschar excision in patients with massive full-thickness burns. This technique is helping both, in the acute stage to cover large raw areas as well as in reducing the amount of donor sites taken from the patient and improving cosmesis associated with an improved quality of life in severely burned patients. The challenge that remains is to make this science economically viable for those who need it most. The scope of surgery too has changed from covering chronic burn wounds and treating disfiguring deformities to management of the acute burn wound. Thus escharectomy, tangential excision, meshed skin grafting, micro-skin grafting, and meek grafting are all invaluable arsenals of our armamentarium. Softened freeze-dried glutaraldehyde-preserved skin, chlorhexidine-alcohol refrigerated porcine skin, and frozen amniotic membrane are all effective as burn dressings. The reconstruction in the burn patient is often a long process requiring multiple procedures and stages. It demands a stepwise and prioritized approach, aiming at both maximum function as well as optimal appearance. With better anaesthetic and critical care support surgeons do not hesitate to climb up the reconstructive ladder and microsurgical reconstructions for the sake of better function and cosmesis are becoming more and more common. Rehabilitation of burn patients to effectively reintegrate them in the society as productive members has been a Herculean task, which is still ongoing. Vocational training has rendered these patients the skills and developmental improvements that are truly outstanding, leading to their return as useful and productive members of their family. The devastating effects of burns are long lasting at both an individual and societal level. These impacts are compounded in resource-poor settings, where the human and material resources necessary to deal with this complex public health problem are thinly distributed over a large population base. A structured and comprehensive approach to burn care must be applied to these resource-poor settings in order to improve outcomes. A combination of improved management and prevention strategies has resulted in important declines in morbidity and mortality in the developed world. Unfortunately, without adequate resources in first-aid, acute surgical management and rehabilitation facilities, patients that do survive their burn injuries in developing countries often have poor, disfiguring and disabling long-term outcomes. Burn care truly deserves a helping hand from both governmental and non-governmental sources and philanthropists should come up with a modestly funded burn care strategy for the developing world.

Functional Properties of Grass Carp (Ctenopharyngodon Idella), Nile Perch (Lates Niloticus) and Nile Tilapia (Oreochromis Niloticus) Skin Hydrolysates

The influence of using an endo-peptidase (alcalase) on the functional properties of hydrolysis products from Nile perch, Grass carp, and Nile tilapia skin was studied. Reaction conditions were controlled at pH 8.25, 60°C, and the enzyme was added on the basis of standard activity units at an enzyme to substrate ratio of 1.7 g/100 g protein. The reaction was allowed to proceed for 85 min and enzyme was inactivated by heat. The soluble protein fractions were recovered and lyophilized. All freeze-dried fish skin hydrolysates powders had a light yellow color and contained up to 90% protein. Nitrogen solubility varied from 95.93 to 98.72% and was not significantly different at 5% probability level. The water and oil holding capacities of the skin hydrolysates were good in the range of 2.8 to 3.2 mL/g and 3.4 to 3.8 mL/g, respectively. Emulsification capacity varied from 11.3 to 21 mL/0.5 g with Nile perch skin hydrolysate having the highest score while Nile tilapia skin hydrolysate was the lowest. Grass carp skin hydrolysate was not able to form stable foam, unlike the Nile perch and Nile tilapia skin hydrolysate. Alcalase treated freshwater fish skin exhibited satisfactory functional properties hence may play an important role as an ingredient in the food and pharmaceutical industry.

Effects of solvent and temperature on pressurized liquid extraction of anthocyanins and total phenolics from dried red grape skin.

Pressurized liquid extraction (PLE) was used to extract anthocyanins from the freeze-dried skin of a highly pigmented red wine grape with six solvents at 50 degrees C, 10.1 MPa, and 3 x 5 min extraction cycles. Temperature (from 20 to 140 degrees C in 20 degrees C increments) effects on anthocyanin recovery by acidified water and acidified 60% methanol were also studied. Acidified methanol extracted the highest levels of total monoglucosides and total anthocyanins, whereas the solvent mixture (40:40:20:0.1 methanol/acetone/water HCl) extracted the highest levels of total phenolics and total acylated anthocyanins. Acidified water extracts obtained by PLE at 80-100 degrees C had the highest levels of total monoglucosides, total acylated anthocyanins, total anthocyanins, total phenolics, and ORAC values. Acidified methanol extracts obtained by PLE at 60 degrees C had the highest levels of total monoglucosides and total anthocyanins, whereas extracts obtained at 120 degrees C had the highest levels of total phenolics. High-temperature PLE (80-100 degrees C) using acidified water, an environmentally friendly solvent, was as effective as acidified 60% methanol in extracting anthocyanins from grape skins.

Determination of residues of oxolinic acid and flumequine in freeze-dried salmon muscle and skin by HPLC with fluorescence detection

A procedure for the determination of residues of oxolinic acid (OA) and flumequine (FLU) in freeze-dried salmon muscle with attached skin, using reversed-phase high-performance liquid chromatography, is described. OA and FLU were extracted by a solid-liquid extraction procedure: after addition of hydrochloric acid, extraction used successively ethyl acetate, sodium hydroxide and chloroform. Liquid chromatography was performed on a 5 μm PuroSpher RP-18E® cartridge using acetonitrile and 0.02 M aqueous orthophosphoric acid solution as mobile phase, with fluorescence detection. The performance of the method was established by spiking tissues with OA and FLU before the freeze-drying step. The method was linear over the concentration range 50–2000 ng/g freeze-dried tissue. Limits of detection and quantitation were 3.2 and 16 ng/g wet weight tissue respectively both for OA and FLU. Mean extraction recoveries of OA and FLU from freeze-dried tissue were 85.5 and 85.2% respectively. The method is suitable as a regulatory one for determination of residues of OA and FLU in freeze-dried salmon tissue.

Antimicrobial peptides from the skin of the Japanese mountain brown frog, Rana ornativentris.

Six peptides with antimicrobial activity were isolated from an extract of freeze-dried skin of the Japanese mountain brown frog Rana ornativentris. Two structurally related peptides (brevinin-20a GLFNVFKGALKTAGKHVAGSLLNQLKCKVSGGC, 11 nmol/g dried tissue, and brevinin-20b GIFNVFKGALKTAGKHVAGSLLNQLKCKVSGEC, 170 nmol/g) belong to the brevinin-2 family, previously identified in Asian and European, but not North American, Ranid frogs. Four peptides (temporin-10a FLPLLASLFSRLL.NH2, 13 nmol/g; temporin-10b FLPLIGKILGTI L.NH2, 350 nmol/g; temporin-10c FLPLLASLFSRLF.NH2, 14 nmol/g; and temporin-10d FLPLLASLFSGLF.NH2, 8 nmol/g) are members of the temporin family first identified in the European common frog Rana temporaria but also found in the skins of North American Ranids. The brevinin-2 peptides showed broad-spectrum activity against the gram-positive bacterium, Staphylococcus aureus, the gram-negative bacterium, Escherichia coli and the yeast Candida albicans, whereas the temporins showed potent activity only against S. aureus. The brevinins and temporins belong to the class of cationic antimicrobial peptides that adopt an amphipathic alpha-helical conformation but it is significant that temporin-10d, which lacks a basic amino acid residue, is still active against S. aureus (minimum inhibitory concentration=13 microM compared with 2 microM for temporin-10a). This suggests that strong electrostatic interaction between the peptide and the negatively charged phospholipids of the cell membrane is not an absolute prerequisite for antimicrobial activity.

Fourier Transform Raman Spectroscopy of 15th Century Mummies from Qilakitsoq, Greenland

To investigate molecular changes in mummified skin, near-infrared Raman spectroscopy was applied to the skin obtained from four mummies found in Qilakitsoq in Greenland. The mummies date from AD 1475 (±50 years) and are the oldest preserved bodies in the Arctic region. The spectra of the skin obtained from the different mummies were very similar, but they were distinctly different from those of fresh and freeze-dried contemporary skin. Especially in the spectra of the ancient skin the amide I (1640–1680 cm-1) and amide III (1220–1300 cm-1) bands had very low intensity, indicating loss of protein and/or changes in the secondary protein structure. Similar spectral changes have previously been found in the 5200-year-old skin of the Iceman. This may suggest that most changes in molecular structure take place in a relatively short time after mummification. © 1997 by John Wiley & Sons, Ltd.

The 55-kd keratohyalin granule protein has the same epitope as the 43-kd stratum corneum membrane protein: immunofluorescence and immunoblotting studies using a monoclonal antibody to the 55-kd keratohyalin granule protein

A monoclonal antibody (Ted-R-1) to the keratohyalin granules (KHGs) of the newborn rat epidermis was developed by immunizing mice with a pH 4.7 hematoxylin-stainable fraction of the extract in a 50 mM Tris-HCl buffer, pH 7.3, containing 10 micrograms/ml phenylmethylsulfonyl fluoride (PMSF) from the newborn rat whole epidermis. Using an indirect immunofluorescence technique, Ted-R-1 was located in two places: (a) the KHGs and (b) the cell membrane region of the stratum corneum. Under immunoelectron microscopy, the antigenic materials were located at the periphery of the KHGs. Immunoblotting analyses of the antigenic pH 4.7 fraction, the Tris-HCl extract from the stratum corneum, and the Tris-8M urea extract from freeze-dried whole skin demonstrated that there were 55 kd, 43 kd, and 78 kd polypeptides, respectively, with which Ted-R-1 reacts. No positive spot was seen in the dermal extract. The major amino acids of the 55 kd protein were glycine (24.4%), serine (17.3%), glutamic acid (14.1%), and alanine (7.7%), while the histidine residue was only 3.1%. On the basis of its amino acid composition, the 55 kd proteins is the third component of KHGs - in addition to histidine-rich and cystine-rich proteins.

PIXE Analysis in Different Stages of Psoriatic Skin

Elemental distribution in psoriatic skin varies with the functional state of the keratinocytes, e.g., electrolytes influence cell metabolism and cell proliferation, and trace elements play a crucial role in a great number of enzymes. Elemental distribution in pinpoint lesions, old plaques, and uninvolved skin of 5 psoriatic patients and 4 healthy controls was studied by means of PIXE (proton-induced x-ray emission) analysis. This technique allows the simultaneous detection of elements with an atomic number greater than or equal to 14 along the epidermis and dermis in freeze-dried skin biopsies. Trace elements such as Fe, Cu, and Zn were determined down to a level of 1 ppm. In comparison with uninvolved skin, concentrations of P and K were elevated in psoriatic epidermis. In addition, increased levels of K were correlated with the stage of the psoriatic lesion. Zinc concentrations were significantly elevated in pinpoint lesions. The Zn concentration profiles within the epidermis and upper dermis showed high correlation to the P concentration profiles. Iron levels were decreased in old psoriatic plaques, whereas Cu concentrations varied considerably. In comparison to the controls, Cl concentrations were markedly decreased in the dermis of involved and uninvolved psoriatic skin, whereas epidermal Cl levels were unaffected. As high K levels prevent the Ca-induced differentiation of keratinocytes, high K levels may be the cause of the high cell differentiation in psoriatic skin. Elevated DNA- and RNA-polymerases might be the cause of elevated Zn levels in pinpoint lesions.

Comparison of fresh autogenous and freeze-dried allogenic skin for mandibular vestibuloplasty

Comparison of fresh autogenous and freeze-dried allogenic skin for mandibular vestibuloplasty

Freeze‐Dried Skin Allografts: A Human Clinical and Histological Study

Free gingival autografts still remain the most predictable method for creating attached gingiva. However, a need exists for sources of connective tissue other than the patient's own connective tissue. Yukna et al. have proposed freeze-dried skin (FDS) allografts as a substitute for gingival autografts. The purpose of the present study was to evaluate FDS allografts on humans, clinically and histologically. Ten FDS allografts and two free gingival (FG) autografts were performed on ten patients, two of them receiving both FDS and FG grafts. Clinical measurements were made before surgery, immediately after surgery and then 2 and 6 months after surgery. After 4 and 24 months, biopsies were performed on both FG and FDS grafts. Routine histologic staining was done. The results show a mean gain of attached gingiva of 0.32 mm with a range from 0 to 0.8 mm. Compared biopsies sections of FDS allografts and FG autografts show that the tissue obtained with the FDS allografts do not display the histomorphologic pattern of attached gingiva. The FDS allografts do not seem to represent a good substitute to free gingival autografts. However, they might be used successfully as a biological bandage.

Evaluation of the immunogenicity of freeze-dried skin allografts in humans: cell-mediated response.

PATIENTS RECEIVING freeze-dried skin (FDS) allografts were evaluated for cell-mediated response by a lymphocytotoxicity test. Eleven patients received single or multiple FDS allografts from a donor typed for human leukocyte antigen (HLA). Heparinized blood samples were drawn before the procedure and at 2, 4, and 8 weeks after grafting. Mononuclear cells were isolated by Ficoll-Hypaque gradients. Cell-mediated lymphocytotoxicity tests were conducted, with mononuclear cells from the FDS allograft recipients used as effector cells. Phytohemagglutinin-stimulated blast target cells of the same HLA type as that of the skin donor were labeled with chromium 51. Lympholysis was evaluated by measuring the amount of 51Cr release after 18 hours' incubation with target/effector cell ratios of 1:100, 1:50, and 1:25. Negative control wells contained target cells alone. Positive control wells contained target cells and effector cells sensitized in vitro against the target cells. Viability of effector cells was tested by trypan-blud dye exclusion and response to phytohemagglutinin. None of the experimental blood samples showed 51Cr release significantly greater than shown by the negative controls. Our findings of no cell-mediated response and the findings of a previous study showing no production of anti-HLA antibody in response to FDS allografts indicate that allogeneic FDS is an immunologically safe material for use in periodontal surgical procedures.

Evaluation of the immunogenicity of freeze-dried skin allografts in humans.

A study was undertaken to evaluate the antigenic potential of allogeneic freeze-dried skin (FDS) when used as a grafting material and to examine the clinical results when FDS is used for the correction of mucogingival problems in humans. FDS was obtained from an HLA-tissue-type donor according to Navy Tissue Bank protocol. Allogeneic FDS grafts were performed to correct mucogingival problems. In addition, periosteal fenestration was used in conjunction with approximately one-half of the grafts. Documentation with a standardized periodontal probe consisted of clinical measurements taken preoperatively and 8 weeks postoperatively. Blood samples were drawn prior to and at 1, 2, 3, 5, and 8 weeks postsurgically. Testing of blood samples for anti-HLA antibody was performed by use of a modified microcytotoxicity assay. Thirty-one patients received 36 FDS grafts involving 148 tooth sites. The FDS allografts resulted in a significant (P < 0.001) increase in the width of attached-type tissue (mean gain = 3.80 mm). Clinically, there was no sign of graft rejection, and serologically, all serum samples tested were nagative for the presence of anti-HLA antibody. FDS grafts used for the treatment of mucogingival problems in humans are nonimmunogenic as evaluated for anti-HLA antibody. The surgical procedure performed using FDS as a graft material resulted in a significant increase in the width of attached-type tissue.

Comparative clinical evaluation of freeze‐dried skin allografts and autogenous gingival grafts in humans*

Freeze-dried skin (FDS) allografts and fresh autogenous free gingival grafts (FGG) were placed in five paired sites in four patients. Both types of grafts healed uneventfully. Evaluation over a 1-year period demonstrated no statistically or clinically significant differences between the FDS and FGG when: a) increase in the width of keratinized gingiva, b) decrease in recession, c) decrease in sulcus depth, k) gain in attachment level, or e) percent shrinkage of the graft were compared. It was concluded that FDS performs essentially similar to FGG in the correction of mucogingival problems, and has the advantages of decreased patient morbidity (no donor site) and availability of abundant amounts of graft material when needed.

Evaluation of the Use of Freeze‐dried Skin Allografts in the Treatment of Human Mucogingival Problems

The purpose of this investigation was to evaluate the clinical responses to freeze-dried skin allografts when used to correct mucogingival problems in humans. FDS was obtained by the NMRI Tissue Bank after processing by standard procedures. The recipient sites were prepared using the standard partial thickness dissection with apical periosteal separation. Rehydrated FDS was sutured into place to completely cover the recipient sites. Routine postoperative procedures were performed. Evaluations were made by means of calibrated periodontal probe measurements, Kodachromes, and radiographs preoperatively and up to 12 months postoperatively. Fourteen patients received 24 FDS allografts over a total of 56 tooth sites. All treated teeth showed a substantial increase (greater than 2 mm) in the width of the zone of keratinized gingiva at all postoperative time periods (2.88 +/- 1.57 mm at 12 months). Other findings included a mean gain of attachment of 0.40 +/- 1.08 mm, a mean decrease in recession of 0.12 +/- 0.91 mm, and mean decrease in pocket depth of 0.33 +/- 0.53 mm. Patient comfort was good, and there were no untoward sequelae, even in patients receiving multiple grafts. This study indicates that FDS allografts are an acceptable and beneficial material for treatment of mucogingival problems in humans.

The use of allogeneic freeze-dried skin grafts in the oral environment: A clinical and histologic evaluation

Allogeneic freeze-dried skin has been used for many years in burn therapy. It can be stored indefinitely at room temperatures and is nonimmunogenic. It has potential use in the oral cavity where there is a limited supply of donor tissue or where the creation of a donor area wound is undesirable. Allogeneic freeze-dried skin was sutured into place over connective-tissue-bed recipient sites in the oral cavities of Macaca mulatta monkeys. Block sections were obtained at 0, 1, 4, 7, 21, 180, and 360 days. Clinical evaluation showed an enhanced uncomplicated healing sequence. Histologic examination revealed a biologic acceptance and incorporation of the collagen fibers of the graft tissue by the host, along with a postoperative reduction in inflammatory and osteoclastic response.

Absence of antigenicity in freeze-dried skin allografts

Summary Studies performed to elucidate differences between grafts of different types of preserved skin have revealed that freeze-dried skin fails to stimulate immunological reactivity in graft recipients. This finding is unique to the freezedrying process and is not due to lack of association of graft with host.

Stored skin homografts in extensivelv burned patients.

This report of 50 extensively burned patients treated with stored split-thickness skin homografts is a result of a clinical research effort which began in 1951. All of the skin homografts in this study were procured, processed, and stored by the Tissue Bank * of the Naval Medical School. This investigation presents data which permit a rough comparison between the mortality rate of burned patients who were treated with fresh1,2and those treated with stored skin homografts. At the outset it was important to determine whether or not stored skin, viable or nonviable, could approximate the clinical take and remain as a biological dressing in a manner which was the equivalent of the fresh homograft. In the instance of the stored viable skin, this did not appear to be questionable, and indeed it was not. In the instance of the known nonviable freeze-dried skin, this question was paramount. In the very