Calf Skin Research Articles

Maximal local thermal hyperemia in able‐bodied and spinal cord injured humans (677.10)

Paraplegic spinal cord injury alters local thermal hyperemic responses. We hypothesized that a similar effect would be observed in tetraplegia. 6 able‐bodied (AB), 5 paraplegic (PARA), and 5 tetraplegic (TETRA) age and gender matched subjects participated. Skin blood flow (SkBF) was monitored by laser‐Doppler flowmetry (LDF) from forearm and calf sites. Local skin temperature (Tloc) was controlled with combined LDF/local heating probe holders. Tloc was measured with thermocouples placed between the probe holders and the skin. Mean arterial pressure (MAP) was recorded by Finapres. After instrumentation, Tloc was increased slowly from normothermic levels to 42°C and maintained at that temperature for at least 25 minutes to cause maximal local vasodilation. Cutaneous vascular conductance was calculated as CVC=LDF/MAP and analyzed by ANOVA. Results: At Tloc=42°C, CVC measured from forearm skin was 47±7mV in AB, 49±8mV in PARA, and 42±8mV in TETRA. These responses did not statistically differ among groups. At Tloc=42°C, CVC measured from calf skin was 26±7mV in AB, 50±8mV in PARA, and 42±8mV in TETRA. These responses differed between AB and PARA (p=0.04) and tended to differ between AB and TETRA (p=0.15). Within groups, forearm responses tended to be greater than calf responses in AB (p=0.06), but not in PARA and TETRA groups. We conclude that spinal cord injury alters maximal SkBF levels in the calf, but not the forearm. In AB persons, maximal SkBF tends to be lower in the calf than the forearm (supported by NIH Grant R03NS083961).Grant Funding Source: Supported by NIH R03NS083961

Purification and Characterization of Pepsin-Solubilized Collagen from Skin of Sea Cucumber Holothuria parva

Pepsin-solubilized collagen (PSC) was extracted from the skin of sea cucumber Holothuria parva and was fractionally characterized. The PSC from H. parva skin consisted of three α1 chains (α1)3, in contrast to calf skin collagen type I with two α1 and one α2 chains (α1)2α2 with approximately 130kDa each. The maximum transition (Tm) and denaturation temperature (Td) of PSC were determined to be 46.94 and 32.5°C, respectively. The amino acid composition analysis revealed that glycine, proline, alanine, and hydroxyproline were the abundant amino acids available in extracted PSC. The results showed that the isolated collagen from H. parva has some similar characteristics to previously reported collagens used in food and pharmaceutical industries.

Effect of lower body compression garments on hemodynamics in response to running session.

Purpose. Compression garments are often worn during exercise and allegedly have ergogenic and/or physiological effects. In this study, we compared hemodynamics and running performance while wearing compression and loose-fit breeches. We hypothesized that in neutral-warm environment compression breeches impair performance by diminishing body cooling via evaporative sweat loss and redistributing blood from active musculature to skin leading to a larger rise in body temperature and prolonging recovery of hemodynamics after exercise. Methods. Changes in hemodynamics (leg blood flow, heart rate, and blood pressure during orthoclinostatic test), calf muscle tissue oxygenation, and skin and core temperatures were measured in response to 30 min running (simulation of aerobic training session) followed by maximal 400 m sprint (evaluation of running performance) in recreationally active females (25.1 ± 4.2 yrs; 63.0 ± 8.6 kg) wearing compression or loose-fit breeches in randomized fashion. Results. Wearing compression breeches resulted in larger skin temperature rise under the garment during exercise and recovery (by about 1°C, P < 0.05; statistical power > 85%), while core temperature dynamics and other measured parameters including circulation, running performance, and sensations were similar compared to wearing loose-fit breeches (P > 0.05). Conclusion. Compared with loose-fit breeches, compression breeches have neither positive nor negative physiological and performance effects for females running in thermoneutral environment.

Influence of Cold-Water Immersion on Limb and Cutaneous Blood Flow after Exercise

This study aimed to determine the influence of cold (8°C) and cool (22°C) water immersion on femoral artery and cutaneous blood flow after exercise. Twelve men completed a continuous cycle exercise protocol at 70% peak oxygen uptake until a core temperature of 38°C was attained. Subjects were then immersed semireclined into 8°C or 22°C water to the iliac crest for 10 min or rested. Rectal and thigh skin temperature, deep and superficial muscle temperature, thigh and calf skin blood flow (laser Doppler flowmetry), and superficial femoral artery blood flow (duplex ultrasound) were measured before and up to 30 min after immersion. Indices of vascular conductance were calculated (flux and blood flow/mean arterial pressure). Reductions in rectal temperature were similar (0.6°C-0.7°C) in all three trials (P = 0.38). The mean ± SD thigh skin temperature during recovery was 25.4°C ± 3.8°C in the 8°C trial, which was lower than the 28.2°C ± 1.4°C and 33.78°C ± 1.0°C in the 22°C and control trials, respectively (P < 0.001). Recovery muscle temperature was also lowest in the 8°C trial (P < 0.01). Femoral artery conductance was similar after immersion in both cooling conditions and was lower (∼55%) compared with the control condition 30 min after immersion (P < 0.01). Similarly, there was greater thigh (P < 0.01) and calf (P < 0.05) cutaneous vasoconstriction during and after immersion in both cooling conditions relative to the control condition. Colder water temperatures may be more effective in the treatment of exercise-induced muscle damage and injury rehabilitation by virtue of greater reductions in muscle temperature and not muscle blood flow.

Correlation of skeletal muscle blood oxygenation level‐dependent MRI and skin laser doppler flowmetry in patients with systemic sclerosis

To investigate the origin of skeletal muscle BOLD MRI alterations in patients with systemic sclerosis (SSc) by correlating BOLD MRI T2* signal of calf muscles with microcirculatory blood flow of calf skin measured by laser Doppler flowmetry (LDF). BOLD MRI (3T) and LDF measurements were performed in 12 consecutive SSc patients (6 women, 6 men; mean age 54.0 ± 10.0 years) and 12 healthy volunteers (4 men, 8 women; mean age 44.7 ± 13.1 years). For both modalities, the same cuff compression paradigm at mid-thigh level was used. LDF datasets were acquired using a PeriScan PIM II Imager (Perimed AB, Stockholm, Sweden) at the upper calf corresponding to the level of MR imaging. Cross-correlations of BOLD and LDF signal intensity changes depending on time lags between both time series were calculated. Maximal cross-correlations of BOLD T2* and LDF measurements were calculated as 0.93 (healthy volunteers) and 0.94 (SSc patients) for a BOLD time lag of approximately 10 s. Key parameter analysis suggested that in contrast to hyperemic BOLD signal loss at maximum value in SSc patients, ischemic T2* decrease cannot be explained by differences of tissue perfusion. Skeletal muscle BOLD T2* signal in SSc patients is closely correlated with changes of microperfusion as detected by LDF.

Treatment of Crowe type IV developmental dysplasia of the hip with total hip arthroplasty after lirnb-lengthing with external fixator

Objective To retrospectively analyze the methods and clinical outcome to treat Crowe type IV developmental dysplasia of the hip （DDH） in young adults with total hip arthroplasty （THA） after limb-lengthing with external fixator. Methods From October 2007 to January 2012, 12 patients with unilateral Crowe type IV DDH were treated with two-staged surgical method in our department. There were 2 males and 10 females with an average age of 25.7 years （range, 18-35 years）. In the first stage, the patients underwent soft tissue relaxation and iliofemoral distraction with use of an external fixator for 10-17 days. There were 1-2 cm distraction at the first time and 3-5 mm daily distraction. When the femoral head was distracted to the level of anatomical position, the second stage-THA was performed. All patients underwent uncemented prosthesis with bulk femoral head autograft for acetabular reconstruction. The acetabular cup was placed in the anatomical position in every patient. Shorten- ing femoral osteotomies were not required. Results The mean time of first operation was 35.2±3.6 min, and hospital stay was 13.3＋1.6 days. The mean time of second operation was 77.3±12.4 rain, and hospital stay was 9.2±2.5 days. The average follow-up was 13.6±3.2 months, limb-length discrepancy was 5.6±1.5 cm on average preoperatively and 0.5±0.2 cm on average postopera- tively. The Harris hip score was increased from 45.7±2.6 preoperatively to 92.3-＋3.3 postoperatively. All of the cases had acquired good hip and knee function. No patient suffered pin-site infection, hip joint infection, prosthesis loosening or deep vein thrombo- sis in our research. Transient nerve palsy occurred during the leg limb lengthening in 3 cases; calf skin numbness after THA oc- curred in 5 cases. Conclusion For the Crowe type IV DDH in young adults, normal limb length can be restored nearly and avoid nerve injury via continuously limb-lengthing with external fixator before THA. This method can get precise results, improve limb function significantly and have fewer complications. Key words: Arthroplasty, replacement, hip; Bone diseases, developmental; Bone lengthening

Beta blockers for peripheral arterial disease

Beta (β) blockers are indicated for use in coronary artery disease (CAD). However, optimal therapy for people with CAD accompanied by intermittent claudication has been controversial because of the presumed peripheral haemodynamic consequences of beta blockers, leading to worsening symptoms of intermittent claudication. This is an update of a review first published in 2008. To quantify the potential harmful effects of beta blockers on maximum walking distance, claudication distance, calf blood flow, calf vascular resistance and skin temperature when used in patients with peripheral arterial disease (PAD). For this update, the Cochrane Peripheral Vascular Diseases Group Trials Search Co-ordinator searched the Specialised Register (last searched March 2013) and the Cochrane Central Register of Controlled Trials (CENTRAL, The Cochrane Library, 2013, Issue 2). Randomised controlled trials (RCTs) evaluating the role of both selective (β1) and non-selective (β1 and β2) beta blockers compared with placebo. We excluded trials that compared different types of beta blockers. Primary outcome measures were claudication distance in metres, time to claudication in minutes and maximum walking distance in metres and minutes (as assessed by treadmill).Secondary outcome measures included calf blood flow (mL/100 mL/min), calf vascular resistance and skin temperature (ºC). We included six RCTs that fulfilled the above criteria, with a total of 119 participants. The beta blockers studied were atenolol, propranolol, pindolol and metoprolol. All trials were of poor quality with the drugs administered over a short time (10 days to two months). None of the primary outcomes were reported by more than one study. Similarly, secondary outcome measures, with the exception of vascular resistance (as reported by three studies), were reported, each by only one study. Pooling of such results was deemed inappropriate. None of the trials showed a statistically significant worsening effect of beta blockers on time to claudication, claudication distance and maximal walking distance as measured on a treadmill, nor on calf blood flow, calf vascular resistance and skin temperature, when compared with placebo. No reports described adverse events associated with the beta blockers studied. Currently, no evidence suggests that beta blockers adversely affect walking distance, calf blood flow, calf vascular resistance and skin temperature in people with intermittent claudication. However, because of the lack of large published trials, beta blockers should be used with caution, if clinically indicated.

Effect of Stemona spp. against Rhipicephalus microplus

Stemona plants have been traditionally used against various insects in Thailand and Southeast Asian countries. The acaricidal efficacy of 9 species of Stemona grown in Thailand was evaluated against dairy cattle tick, Rhipicephalus microplus using adult immersion test and in vivo evaluation on infested calves. From the ten Stemona root extracts used in this study, S. collinsiae of a concentration 250 mg/ml possessed the highest activity. In vivo study revealed that S. collinsiae extract could significantly reduce the attached ticks on calf skin compared to the control and promoted not significantly different efficacy from flumethrin, a common pyrethroid used in dairy farms. No side effect was found on calves during the experiment. The results confirmed traditional use of S. collinsiae as a better source of insecticide than other species and could be used as guidance for further development of S. collinsiae as a herbal acaricide.

Characterization of Acid-soluble Collagen from the Skin of Hammerhead Shark (Sphyrna lewini )

The characteristics of the acid-soluble collagen (ASC) from the skin of hammerhead shark (Sphyrna lewini) (ASC-H) were investigated and compared with those of calf skin collagen (CSC). ASC-H with a yield of 4.23 ± 0.54% (based on the wet weight of skins) contained Gly (227 residues/1,000 residues) as the major amino acid and had imino acids of 205 residues/1,000 residues. Amino acid composition, sodium dodecyl sulfate polyacrylamide gel electrophoresis pattern and Fourier transform spectroscopy (FTIR), confirmed that ASC-H was mainly composed of type I collagen. The peptide map of ASC-H was different from that of CSC, suggesting the differences in amino acid sequences and conformation. Td of ASC-H was 16.89C, which was similar to those of cold-water fishes but significantly lower than those of tropical fish species and mammals. ASC-H exhibited high solubility in pH (1-4) and low NaCl concentrations (≤3%). In addition, the lyophilized collagen displayed loose, fibrous and porous ultrastructure. Practical Applications At present, large quantities of by-products, accounting for 50–70% of the original raw material, are generated during the process of aquatic products processing industry. Therefore, optimal use of these by-products is a promising way to protect the environment and produce value-added products to increase the revenue of fish processors. Recently, many scientists have focused their interests on isolation and characterization of collagens extracted from skins of aquatic organisms. However, no information regarding acid-soluble collagen (ASC) from skins of Hammerhead shark (Sphyrna lewini) has been reported. The aim of this study was to isolate and characterize ASC from skin of Hammerhead shark in comparison with type I collagen from calf skin (CSC). Therefore, the collagen extracted from the skin of hammerhead shark could bring considerably economic benefits as a substitute for mammalian collagen.

Characterization of Acid-Soluble Collagen From Bone of Pacific Cod (Gadus macrocephalus)

Acid-soluble collagen (ASC) was isolated from Pacific cod (Gadus macrocephalus) bone. The ASC was rich in glycine. The amount of imino acid was lower than that of calf skin collagen, as was the transition temperature (48.6°C). Electrophoresis revealed two different α chains (α1 and α2), β-component, and γ-component. Fourier transform infrared spectroscopy measurement showed that ASC was in triple-helix structure. ASC had a solubility greater than 90% in a very acidic pH range (pH 1–4), and the solubility decreased with increasing NaCl concentrations up to 3%. Lyophilized ASC had a network ultrastructure with lace-like fibers, similar to calf skin collagen sponge.

Effects of two substrate types on the behaviour, cleanliness and thermoregulation of dairy calves

The use of river stones as a substrate to rear calves on is a management practice that is becoming more common in parts of New Zealand where more traditional substrate types, such as sawdust, can be difficult and/or expensive to obtain. A study was conducted to compare the behaviour, cleanliness, thermoregulation and weight gain of calves reared on river stones or sawdust. At 1wk of age, 40 calves were moved into four pens with floors covered with river stones (RS: n=5 calves/pen) or four pens with floors covered with sawdust (SD: n=5 calves/pen). The river stones were approximately 3cm in length and rounded with a smooth surface. Calf behaviour, body weight, cleanliness and skin surface temperature were recorded at 1 and 5wks of age. Behaviour was video recorded continuously for 24h; the time calves spent lying, standing, walking and running were estimated using 1-min instantaneous scan sampling. In addition, during this period the frequency and duration of lying and play behaviour was recorded continuously for 10h during daytime hours. Skin surface temperature of the calves was recorded continuously using iButtons® and floor surface temperature was recorded continuously using data-loggers during the first and sixth week of the study period. At 1wk of age, calves reared on sawdust played more; they spent more (P<0.001) time running (0.1 and 0.3±0.04% total observations/24h, RS and SD respectively) and performed more (P<0.005, total observations/10h) head shakes, jumps, kicks and leaps compared with calves reared on river stones. At 5wk of age, calves reared on sawdust spent more (P<0.001) time lying (67.6 and 72.1±1.34% total observations/24h, RS and SD respectively) and performed more (P<0.05, total observations/10h) heading shakes and kicks compared with calves reared on river stones. Skin temperature was higher (P<0.001) for calves reared on sawdust at both ages compared to river stones. There were no treatment differences (P>0.05) in body weight, cleanliness and floor surface temperature. In conclusion, reduced lying and play behaviour in combination with lower skin temperature suggest that calves may find river stones more uncomfortable and/or difficult to move on and that river stones have less insulation properties than sawdust under the conditions studied.

Xiamenmycin Attenuates Hypertrophic Scars by Suppressing Local Inflammation and the Effects of Mechanical Stress

Hypertrophic scarring is a common disease affecting millions of people around the world, but there are currently no satisfactory drugs to treat the disease. Exaggerated inflammation and mechanical stress have been shown to be two main mechanisms of excessive fibrotic diseases. Here we found that a benzopyran natural product, xiamenmycin, could significantly attenuate hypertrophic scar formation in a mechanical stretch-induced mouse model. The compound suppressed local inflammation by reducing CD4+ lymphocyte and monocyte/macrophage retention in fibrotic foci and blocked fibroblast adhesion with monocytes. Both in vivo and in vitro studies found that the compound inhibited the mechanical stress-induced profibrotic effects by suppressing proliferation, activation, fibroblast contraction, and inactivating FAK, p38, and Rho guanosine triphosphatase signaling. Taken together, the compound could simultaneously suppress both the inflammatory and mechanical stress responses, which are the two pivotal pathological processes in hypertrophic scar formation, thus suggesting that xiamenmycin can serve as a potential agent for treating hypertrophic scar formation and other excessive fibrotic diseases.

Characterization of acylated pepsin-solubilized collagen with better surface activity

A new kind of acylated collagen with water solubility and better surface activity was prepared via reaction of pepsin-solubilized calf skin collagen with lauroyl chloride and succinic anhydride in this paper. The equilibrium surface tension and the isoelectric point were 55.92mN/m and 4.93 respectively, suggesting that acylated collagen had surface activity as well as water solubility. Meanwhile, the results of Fourier transform infrared spectroscopy analyses and electrophoresis patterns demonstrated that the triple helix conformation of collagen was not destroyed, but the subunits of acylated collagen shifted to higher molecular weight than those of native collagen. Scanning electron microscope and differential scanning calorimeter measurements revealed that lyophilized acylated collagen exhibited relatively well-distributed pore structure and its denaturation temperature was about 9.0°C higher than that of native collagen. Additionally, the increase of the diameter of the fibrils was observed by atomic force microscopy. Acylated collagen with water solubility and better surface activity might broaden the application of collagen-based materials to cosmetics, drug delivery and pharmacotherapy.

Biochemical and thermo-mechanical analysis of collagen from the skin of Asian Sea bass (Lates calcarifer) and Australasian Snapper (Pagrus auratus), an alternative for mammalian collagen

Australasia has a large fish industry, and fish skin by-products from the processing industry could be used for the commercial production of fish collagen. The aim of this study was to characterize collagen extracted from the Asian sea bass (Australian barramundi) (Lates calcarifer) and snapper (Pagrus auratus) skin as an alternative to mammalian-derived collagen in gelatin products. The acid-soluble fractions of collagen from Asian sea bass and snapper skin were extracted and yielded about 8 and 7.5 % collagen (on a dry weight basis), respectively. The electrophoretic and chromatography patterns indicated that both collagens comprise of α1, α2, α3, and β chains, corresponding to the properties of calf skin collagen type I. Amino acid analysis and peptide mapping of digested collagen suggested differences in their amino acid sequences and collagen primary structure. Fourier transform infrared spectroscopy demonstrated that the helical structure of collagen was completely maintained in Asian sea bass and partially in snapper. Transition temperatures for the completion of the melting process in the two collagen networks were confirmed with differential scanning calorimetry and dynamic oscillatory rheology to be about 29 °C. Zeta potential analysis identified the isoelectric points (pI values) of collagen from Asian sea bass and snapper skin at pH 6.90 and 7.75, respectively. Thus, Asian sea bass and snapper skin could be an important alternative source of collagen to replace mammalian collagen for industrial applications.

Resemblance of Electrospun Collagen Nanofibers to Their Native Structure

Electrospinning is a promising method to mimic the native structure of the extracellular matrix. Collagen is the material of choice, since it is a natural fibrous structural protein. It is an open question how much the spinning process preserves or alters the native structure of collagen. There are conflicting results in the literature, mainly due to the different solvent systems in use and due to the fact that gelatin is employed as a reference state for the completely unfolded state of collagen in calculations. Here we used circular dichroism (CD) and Fourier-transform infrared spectroscopy (FTIR) to investigate the structure of regenerated collagen samples and scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to illuminate the electrospun nanofibers. Collagen is mostly composed of folded and unfolded structures with different ratios, depending on the applied temperature. Therefore, CD spectra were acquired as a temperature series during thermal denaturation of native calf skin collagen type I and used as a reference basis to extract the degree of collagen folding in the regenerated electrospun samples. We discussed three different approaches to determine the folded fraction of collagen, based on CD spectra of collagen from 185 to 260 nm, since it would not be sufficient to obtain simply the fraction of folded structure θ from the ellipticity at a single wavelength of 221.5 nm. We demonstrated that collagen almost completely unfolded in fluorinated solvents and partially preserved its folded structure θ in HAc/EtOH. However, during the spinning process it refolded and the PP-II fraction increased. Nevertheless, it did not exceed 42% as deduced from the different secondary structure evaluation methods, discussed here. PP-II fractions in electrospun collagen nanofibers were almost same, being independent from the initial solvent systems which were used to solubilize the collagen for electrospinning process.

Medial sural perforator plus island flap: A modification of the medial sural perforator island flap for the reconstruction of postburn knee flexion contractures using burned calf skin

Medial sural perforator plus island flap: A modification of the medial sural perforator island flap for the reconstruction of postburn knee flexion contractures using burned calf skin

The Synthesis and Characterization of Nano-hydroxyapatite (nHAP)-g-poly(lactide-co-glycolide)-g-collagen Polymer for Tissue Engineering Scaffolds

ABSTRACTBone grafts, commonly performed to augment bone regeneration from autologous or alleogeneic sources, carry an enormous cost, estimated at upwards of 21 billion dollars per year. Hydroxyapatite (HAP) bio-ceramic has been widely used in clinic as a bone graft substitute material due to its biocompatibility and the similarity of its structure and composition to bone mineral. However, its applications are limited due to its lack of strength and toughness. Researchers have attempted to overcome these issues by combining HAP bio-ceramics into resorbable polymers to improve their mechanical properties. However, poor bonding between the HAP and the polymer caused separation at the polymer-filler interface. To overcome this, short chains of polymers were grafted directly from the hydroxyl groups on the surface of nanocrystalline HAP. Collagens, being the most abundant proteins in the body, and having suitable properties such as biodegradability, bioabsorbability with low antigenicity, high affinity to water, and the ability to interact with cells through integrin recognition, makes them a very promising candidate for the modification of the polymer surface. In this study, a novel method of synthesizing nano-hydroxyapatite (nHAP)-g-poly(lactide-co-glycolide)-g-collagen polymer was introduced. The synthesis process was carried out in several steps. First, poly (lactide-co-glycolide) (PLGA) polymer was directly grafted onto the hydroxyl group of the surface of n-HAP particles by ring-opening polymerization, and subsequently coupled with succinic anhydride. In order to activate the co-polymer for collagen attachment, the carboxyl end group obtained from succinic anhydride was reacted with N-hydroxysuccinimide (NHS) in the presence of dicyclohexylcarbodiimide (DCC) as the cross-linking agent. Finally, the activated co-polymer was attached to calf skin collagen type I, in hydrochloric acid/phosphate buffer solution and the precipitated co-polymer with attached collagen was isolated. The synthesis was monitored by 1H NMR and FTIR spectroscopies and the products after each step were characterized by thermal analysis (TGA and DSC). These composite materials will be tested as potential scaffolds for tissue engineering applications.

Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines to the study of isolated rodent islets. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular signaling pathways and secretory responses. Whereas the method of islet isolation using tissue dissociating enzyme (TDE) preparations has been well established in many laboratories1-4, variations in the consistency of islet yield and quality from any given rodent strain limit the extent and feasibility of primary islet studies. These variations often occur as a result of the crude partially purified TDEs used in the islet isolation procedure; TDEs frequently exhibit lot-to-lot variations in activity and often require adjustments to the dose of enzyme used. A small number of reports have used purified TDEs for rodent cell isolations5, 6, but the practice is not widespread despite the routine use and advantages of purified TDEs for human islet isolations. In collaboration with VitaCyte, LLC (Indianapolis, IN), we developed a modified mouse islet isolation protocol based on that described by Gotoh7, 8, in which the TDEs are perfused directly into the pancreatic duct of mice, followed by crude tissue fractionation through a Histopaque gradient9, and isolation of purified islets. A significant difference in our protocol is the use of purified collagenase (CIzyme MA) and neutral protease (CIzyme BP) combination. The collagenase was characterized by the use of a6 fluorescence collagen degrading activity (CDA) assay that utilized fluorescently labeled soluble calf skin fibrils as substrate6. This substrate is more predictive of the kinetics of collagen degradation in the tissue matrix because it relies on native collagen as the substrate. The protease was characterized with a sensitive fluorescent kinetic assay10. Utilizing these improved assays along with more traditional biochemical analysis enable the TDE to be manufactured more consistently, leading to improved performance consistency between lots. The protocol described in here was optimized for maximal islet yield and optimal islet morphology using C57BL/6 mice. During the development of this protocol, several combinations of collagenase and neutral proteases were evaluated at different concentrations, and the final ratio of collagenase:neutral protease of 35:10 represents enzyme performance comparable to Sigma Type XI. Because significant variability in average islet yields from different strains of rats and mice have been reported, additional modifications of the TDE composition should be made to improve the yield and quality of islets recovered from different species and strains.

Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines to the study of isolated rodent islets. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular signaling pathways and secretory responses. Whereas the method of islet isolation using tissue dissociating enzyme (TDE) preparations has been well established in many laboratories1-4, variations in the consistency of islet yield and quality from any given rodent strain limit the extent and feasibility of primary islet studies. These variations often occur as a result of the crude partially purified TDEs used in the islet isolation procedure; TDEs frequently exhibit lot-to-lot variations in activity and often require adjustments to the dose of enzyme used. A small number of reports have used purified TDEs for rodent cell isolations5, 6, but the practice is not widespread despite the routine use and advantages of purified TDEs for human islet isolations. In collaboration with VitaCyte, LLC (Indianapolis, IN), we developed a modified mouse islet isolation protocol based on that described by Gotoh7, 8, in which the TDEs are perfused directly into the pancreatic duct of mice, followed by crude tissue fractionation through a Histopaque gradient9, and isolation of purified islets. A significant difference in our protocol is the use of purified collagenase (CIzyme MA) and neutral protease (CIzyme BP) combination. The collagenase was characterized by the use of a6 fluorescence collagen degrading activity (CDA) assay that utilized fluorescently labeled soluble calf skin fibrils as substrate6. This substrate is more predictive of the kinetics of collagen degradation in the tissue matrix because it relies on native collagen as the substrate. The protease was characterized with a sensitive fluorescent kinetic assay10. Utilizing these improved assays along with more traditional biochemical analysis enable the TDE to be manufactured more consistently, leading to improved performance consistency between lots. The protocol described in here was optimized for maximal islet yield and optimal islet morphology using C57BL/6 mice. During the development of this protocol, several combinations of collagenase and neutral proteases were evaluated at different concentrations, and the final ratio of collagenase:neutral protease of 35:10 represents enzyme performance comparable to Sigma Type XI. Because significant variability in average islet yields from different strains of rats and mice have been reported, additional modifications of the TDE composition should be made to improve the yield and quality of islets recovered from different species and strains.

Isolation and characterization of pepsin‐solubilized collagen from the integument of sea cucumber (Stichopus vastus)

Sea cucumber (Stichopus vastus) is considered an underutilized resource, since only its stomach and intestines are eaten raw as salad in a few countries and the remaining parts, especially the integument rich in collagen, is discarded. Hence a valuable by-product having potential nutraceutical and pharmaceutical applications is wasted. In the present investigation, pepsin-solubilized collagen (PSC) from the integument of S. vastus was isolated, purified and characterized. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis showed that the purified collagen was of type I, consisting of three α1 chains of approximately 122 kDa each. The peptide map of PSC digested by V8 protease was different from that of calf skin type I collagen. Fourier transform infrared spectroscopy revealed that the triple helical structure was well preserved in isolated collagen. The denaturation temperature of PSC was 21.23 °C and showed good gel-forming capability at pH 6.5 and 300 mmol L⁻¹ NaCl. It is inferred that the collagen isolated from S. vastus integument has potential for use as an alternative to land-based mammalian collagen in food, nutraceuticals and pharmaceutical industries.