Commercial Collagen Research Articles

Biomimetic microgels for articular cartilage regeneration. A minipig knee model

The aim of this study is to explore, with an in vivo model, the regeneration of articular cartilage that can be obtained with a strategy that combines the stimulation of the subchondral bone with the implantation of a biomimetic microgel at the site of the cartilage defect. The microgel consists of an agglomerate of two types of microspheres: ones rigid, made of a biodegradable polyester, and the other ones composed of a polymeric network of gelatin and hyaluronic acid that encapsulate platelet-rich plasma (PRP) obtained from circulating autologous blood. A defect of 7 mm in diameter and full depth was performed in the in the articular cartilage at the trochlea of the animal knee, using a minipig model. Microdrilling was performed in the underlying bone and the defect was filled with the microgel in which the platelets it contains were previously activated with calcium chloride. The whole defect containing the microgel was covered with a synthetic membrane to avoid the release of microspheres. The results of the in vivo follow-up of the experiment, the histological analysis and the mechanical measurements of the regenerated tissue 9 months after implantation were compared with those of a group of animals in which only microdrilling was performed and the defect was covered with the membrane and with another group in which a clinical use commercial collagen mesh was implanted. The mean value of the elastic modulus of the newly formed tissue was not significantly different from that of the native tissue in the case of microgel implantation while its content of GAGs, was significantly better than in the other groups.

Comparison of Commercial Collagen and Marine Collagen: Efficiency and Effects on the Formation of EDC/NHS-Crosslinked Membranes

AbstractThis study investigates the extraction and characterisation of collagen derived from fish skin and scales, the collagens obtained were used to produce membranes which were subsequently cross-linked by EDC/NHS. A comprehensive investigation of their microstructure, composition, mechanical properties, swelling, degradation, thermal properties and a 24 h cell viability test was carried out. The results showed that the yields of collagen from skin and fish scales were 25.14 ± 2.90 and 29.26 ± 2.53%, respectively. Mechanical stress tests showed that membranes made from fish skin collagen and commercial collagen had comparable load-bearing capacities before failure. The Young’s modulus of the membranes was 9.45 MPa ± 1.77 for commercial collagen, 5.76 MPa ± 0.74 for fish skin collagen and 2.43 MPa ± 0.78 for fish scale collagen, this similarity was maintained in degradation tests and at denaturation temperature (75 °C).These results demonstrate the viability of collagen derived from marine by-products as an effective and efficient alternative for biomedical and tissue engineering applications.

Arginine-loaded globular BSAMA/fibrous GelMA biohybrid cryogels with multifunctional features and enhanced healing for soft gingival tissue regeneration

Mucogingival surgery has been widely used in soft gingival tissue augmentation in which autografts are predominantly employed. However, the autografts face grand challenges, such as scarcity of palatal donor tissue and postoperative discomfort. Therefore, development of alternative soft tissue substitutes has been an imperative need. Here, we engineered an interconnected porous bovine serum albumin methacryloyl (BSAMA: B, as a drug carrier and antioxidant)/gelatin methacryloyl (GelMA: G, as a biocompatible collagen-like component)-based cryogel with L-Arginine (Arg) loaded as an angiogenic molecule, which could serve as a promising gingival tissue biohybrid scaffold. BG@Arg cryogels featured macroporous architecture, biodegradation, sponge-like properties, suturability, and sustained Arg release. Moreover, BG@Arg cryogels promoted vessel formation and collagen deposition which play an important role in tissue regeneration. Most interestingly, BG@Arg cryogels were found to enhance antioxidant effects. Finally, the therapeutic effect of BG@Arg on promoting tissue regeneration was confirmed in rat full-thickness skin and oral gingival defect models. In vivo results revealed that BG@Arg2 could promote better angiogenesis, more collagen production, and better modulation of inflammation, as compared to a commercial collagen membrane. These advantages might render BG@Arg cryogels a promising alternative to commercial collagen membrane products and possibly autografts for soft gingival tissue regeneration.

PHYSICAL STABILITY AND SAFETY TESTING OF COSMETIC FORMULATIONS CONTAINING COLLAGEN FROM CATFISH (PANGASIUS SP.) SWIM BLADDER

Objective: Collagen is a fibrous protein that confers strength and flexibility on bone and other body tissues, such as the skin and tendons, and is the main component of the extracellular matrix. It is used in cosmetics for its anti-aging activity. The primary source of commercial collagen is pig skin, but its use is imperfect due to halal concerns and the risk of biological contamination. Therefore, this research was conducted to identify alternate sources of collagen derived from fish. This study used the swim bladders of catfish that were not consumed and sought to separate the collagen from and use it in a safe anti-ageing serum for humans. Methods: Three formulations of 1%, 2%, and 3% collagen were developed in the laboratory using experimental methods. The three formulations were then evaluated for physical stability and in vivo irritation. Results: Physical quality testing showed that the 1%, 2%, and 3% formulations remained stable at room, low, and high temperatures. However, in the irritation test, the 2% and 3% formulations resulted in mild erythema after 24, 48, and 72 h under constant conditions. Conclusion: The 1% formula was confirmed stable and safe.

Pharmacokinetic study of local and systemic gentamicin concentrations after subcutaneous implantation of a gentamicin-impregnated collagen sponge in dogs

This study evaluated the pharmacokinetics of commercial gentamicin-impregnated collagen sponges (GICS) applied subcutaneously in dogs. In six healthy beagles, an 11 ×6 cm subcutaneous pocket was created, a folded 10×10 cm GICS was inserted, and saline was injected to mimic a seroma. Wound fluid samples were aspirated, and the gentamicin concentration was determined. Simultaneously, blood samples were collected to evaluate the corresponding systemic gentamicin concentration. All samples were collected before and 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 18, 24, 36, 48, 72, 96, 120, and 168 hours after GICS placement. The local Cmax of gentamicin was reached after 0.5 hours (range, 0.5–1.0 hours) post-implantation in 5/6 dogs at a median concentration of 2053.3 µg/mL (range, 918.0–2791.9 µg/mL). Whitin 24 hours, the local concentration dropped below the MIC for Staphylococcus sp. (4 µg/mL) in 5/6 dogs. Plasma Cmax was achieved at a median of 1.2 hours post-implantation (range, 1.0–2.0 hours) and reached a median concentration of 10.3 µg/mL (range, 8.8–18.03 µg/mL). After 6 hours, the gentamicin concentration in the plasma was below 4 µg/mL in all dogs. The GICS provided a high local concentration of gentamicin in a short time with a local Cmax:MIC ratio of 513:1, largely sufficient to eliminate susceptible bacteria, including methicillin-resistant Staphylococcus pseudintermedius (MRSP) and Pseudomonas sp., in a clinical setting. The repeated administration of saline in the present study seemed to have induced a quicker gentamicin release from the GICS than described in previous studies that typically dealt with "drier" wounds.

Stem-Cell-Regenerative and Protective Effects of Squid (Symplectoteuthis oualaniensis) Skin Collagen Peptides against H2O2-Induced Fibroblast Injury.

Recently, there has been a growing interest in collagen peptides derived from marine sources for their notable ability to protect skin cells against apoptosis induced by oxidants. Therefore, the current study aimed to investigate the fundamental properties of collagen peptides, including their physicochemical, thermal, structural, stem-cell-regenerative, and skin-cell-protective effects, in comparison to commercial collagen peptides. The acid-soluble (ASC) and pepsin-soluble (PSC) collagens exhibited three distinct bands on SDS-PAGE, namely α (α1 and α2), β, and γ chains, confirming a type I pattern. The thermal profiles obtained from TG and DSC analyses confirmed the denaturation of PSC and ASC at temperatures ranging from 51.94 to 56.4 °C and from 52.07 to 56.53 °C, respectively. The purified collagen peptides were analyzed using SDS-PAGE and MALDI-TOF mass spectrometry, revealing a mass range of 900-15,000 Da. Furthermore, the de novo peptide sequence analysis confirmed the presence of the Gly-X-Y repeating sequence in collagen peptides. Collagen peptide treatments significantly enhanced HFF-1 cell proliferation and migration compared to the control group. ELISA results confirmed the potential interactions between collagen peptides and HFF-1 cells through α2β1, α10β1, and α11β1 integrin receptors. Notably, collagen peptide treatment effectively restored the proliferation of HFF-1 cells damaged by H2O2. Consequently, the advantageous characteristics of squid skin collagen peptides highlight their promising role in regenerative medicine.

Human Dermal Decellularized ECM Hydrogels as Scaffolds for 3D In Vitro Skin Aging Models.

Biomaterials play an important role in the development of advancing three dimensional (3D) in vitro skin models, providing valuable insights for drug testing and tissue-specific modeling. Commercial materials, such as collagen, fibrin or alginate, have been widely used in skin modeling. However, they do not adequately represent the molecular complexity of skin components. On this regard, the development of novel biomaterials that represent the complexity of tissues is becoming more important in the design of advanced models. In this study, we have obtained aged human decellularized dermal extracellular matrix (dECM) hydrogels extracted from cadaveric human skin and demonstrated their potential as scaffold for advanced skin models. These dECM hydrogels effectively reproduce the complex fibrillar structure of other common scaffolds, exhibiting similar mechanical properties, while preserving the molecular composition of the native dermis. It is worth noting that fibroblasts embedded within human dECM hydrogels exhibit a behavior more representative of natural skin compared to commercial collagen hydrogels, where uncontrolled cell proliferation leads to material shrinkage. The described human dECM hydrogel is able to be used as scaffold for dermal fibroblasts in a skin aging-on-a-chip model. These results demonstrate that dECM hydrogels preserve essential components of the native human dermis making them a suitable option for the development of 3D skin aging models that accurately represent the cellular microenvironment, improving existing in vitro skin models and allowing for more reliable results in dermatopathological studies.

Preparation and optimization of an eggshell membrane-based biomaterial for GTR applications

ObjectivesGuided Tissue Regeneration (GTR) is a popular clinical procedure for periodontal tissue regeneration. However, its key component, the barrier membrane, is largely collagen-based and is still quite expensive, posing a financial burden to the patients as well as healthcare systems and negatively impacting the patient’s decision-making. Thus, our aim is to prepare a novel biomimetic GTR membrane utilizing a natural biomaterial, soluble eggshell membrane protein (SEP), which is economical as it comes from an abundant industrial waste from food and poultry industries, unlike collagen. Additive polymer, poly (lactic-co-glycolic acid) (PLGA), and a bioceramic, nano-hydroxyapatite (HAp), were added to improve its mechanical and biological properties. MethodsFor this barrier membrane preparation, we initially screened the significant factors affecting its mechanical properties using Taguchi orthogonal array design and further optimized the significant factors using response surface methodology. Furthermore, this membrane was characterized using SEM, EDAX, and ATR-FTIR, and tested for proliferation activity of human periodontal ligament fibroblasts (HPLFs). ResultsOptimization using response surface methodology predicted that the maximal tensile strength of 3.1 MPa and modulus of 39.9 MPa could be obtained at membrane composition of 8.9 wt% PLGA, 7.2 wt% of SEP, and 2 wt% HAp. Optimized PLGA/SEP/HAp membrane specimens that were electrospun on a static collector showed higher proliferation activity of HPLFs compared to tissue culture polystyrene and a commercial collagen membrane. SignificanceFrom the results observed, we can conclude that SEP-based nanofibrous GTR membrane could be a promising, environment-friendly, and cost-effective alternative for commercial collagen-based GTR membrane products.

Janus Membrane with Intrafibrillarly Strontium-Apatite-Mineralized Collagen for Guided Bone Regeneration.

Commercial collagen membranes face difficulty in guided bone regeneration (GBR) due to the absence of hierarchical structural design, effective interface management, and diverse bioactivity. Herein, a Janus membrane called SrJM is developed that consists of a porous collagen face to enhance osteogenic function and a dense face to maintain barrier function. Specifically, biomimetic intrafibrillar mineralization of collagen with strontium apatite is realized by liquid precursors of amorphous strontium phosphate. Polycaprolactone methacryloyl is further integrated on one side of the collagen as a dense face, which endows SrJM with mechanical support and a prolonged lifespan. In vitro experiments demonstrate that the dense face of SrJM acts as a strong barrier against fibroblasts, while the porous face significantly promotes cell adhesion and osteogenic differentiation through activation of calcium-sensitive receptor/integrin/Wnt signaling pathways. Meanwhile, SrJM effectively enhances osteogenesis and angiogenesis by recruiting stem cells and modulating osteoimmune response, thus creating an ideal microenvironment for bone regeneration. In vivo studies verify that the bone defect region guided by SrJM is completely repaired by newly formed vascularized bone. Overall, the outstanding performance of SrJM supports its ongoing development as a multifunctional GBR membrane, and this study provides a versatile strategy of fabricating collagen-based biomaterials for hard tissue regeneration.

Enhanced barrier properties in sweet potato starch films via dual modification by octenyl succinylation and heat moisture treatment for use as plant-based sausage casings.

This study assesses the impact of dual modification [octenyl succinylation (OSA) and heat-moisture treatment (HMT)] of sweet potato starch (SPS) on the physicochemical, mechanical, and permeability properties of SPS film. The intrinsic limitations of starch films, such as sensitivity to high humidity, inferior mechanical properties, and weak barrier capabilities, have restricted their use in sausage casings. Nonetheless, the dual-modified SPS film (OSA@HMT-SPS film) demonstrated significantly reduced solubility (P < 0.05), moisture content, water vapor permeability (WVP), and O2 permeability compared to the SPS film. Furthermore, its flexibility and elasticity, indicated by its elongation at break, was notably superior. When used as sausage casings, the OSA@HMT-SPS film effectively mitigated lipid oxidation in sausages better than both the SPS film and commercial collagen casings, owing to its low O2 permeability. As a result, the OSA@HMT-SPS casing presents significant promise as a plant-based sausage casing alternative.

Assessment of Tilapia Skin Collagen for Biomedical Research Applications in Comparison with Mammalian Collagen.

Collagen is an important material for biomedical research, but using mammalian tissue-derived collagen carries the risk of zoonotic disease transmission. Marine organisms, such as farmed tilapia, have emerged as a safe alternative source of collagen for biomedical research. However, the tilapia collagen products for biomedical research are rare, and their biological functions remain largely unexamined. In this study, we characterized a commercial tilapia skin collagen using SDS-PAGE and fibril formation assays and evaluated its effects on skin fibroblast adhesion, proliferation, and migration, comparing it with commercial collagen from rat tails, porcine skin, and bovine skin. The results showed that tilapia skin collagen is a type I collagen, similar to rat tail collagen, and has a faster fibril formation rate and better-promoting effects on cell migration than porcine and bovine skin collagen. We also confirmed its application in a 3D culture for kidney cells' spherical cyst formation, fibroblast-induced gel contraction, and tumor spheroid interfacial invasion. Furthermore, we demonstrated that the freeze-dried tilapia skin collagen scaffold improved wound closure in a mouse excisional wound model, similar to commercial porcine or bovine collagen wound dressings. In conclusion, tilapia skin collagen is an ideal biomaterial for biomedical research.

Collagen properties of Indonesian local sheepskin isolated using acid and enzymatic methods

Objectives: This study aimed to investigate the physical properties of Indonesian local sheep skin collagen extracted by acid and enzymatic methods. Materials and Methods: Collagen was isolated from Pure Breed Garut Sheep (Ovis aries sp.) skin, 1.5 years old. The skins were obtained from a local slaughterhouse in Cirebon, Indonesia. The solvents used were CH3COOH and three different enzymes: neutrase, alcalase, and bromelain. Results: The highest yields of extracted collagen were bromelain-soluble collagen (BSC), which reached 37.07%. The range of Ph values for all samples started from 4.01 to 4.76. The viscosity values (cP) of acid-soluble collagen (ASC), neutrase-soluble collagen (NSC), alcalase-soluble collagen (LSC), and BSC were 3.42, 3.90, 3.45, and 3.12, respectively. Regarding SDS-PAGE analyses, Garut sheepskin collagen is categorized as collagen type I, which has a molecular weight of about 140.99 to 148.74 kDa for α1 and around 110 to 111.86 kDa for α2. The results of FTIR and DSC analyses for all samples show the same motif with commercial collagen motifs based on the literature. Conclusion: Garut sheep skin has the potential to be an alternative raw material source for producing collagen. Collagen extracted using a combination of CH3COOH and bromelain enzyme showed the most desirable results in almost every characteristic.

Harmonizing Thickness and Permeability in Bone Tissue Engineering: A Novel Silk Fibroin Membrane Inspired by Spider Silk Dynamics.

Guided bone regeneration gathers significant interest in the realm of bone tissue engineering; however, the interplay between membrane thickness and permeability continues to pose a challenge that can be addressed by the water-collecting mechanism of spider silk, where water droplets efficiently move from smooth filaments to rough conical nodules. Inspired by the natural design of spider silk, an innovative silk fibroin membrane is developed featuring directional fluid transportation via harmoniously integrating a smooth, dense layer with a rough, loose layer; conical microchannels are engineered in the smooth and compact layer. Consequently, double-layered membranes with cone-shaped microporous passageways (CSMP-DSF membrane) are designed for in situ bone repair. Through extensive in vitro testing, it is noted that the CSMP-DSF membrane guides liquid flow from the compact layer's surface to the loose layer, enabling rapid diffusion. Remarkably, the CSMP-DSF membrane demonstrates superior mechanical properties and resistance to bacterial adhesion. When applied in vivo, the CSMP-DSF membrane achieves results on par with the commercial Bio-Gide collagen membranes. This innovative integration of a cross-thickness wetting gradient structure offers a novel solution, harmonizing the often-conflicting requirements of material transport, mechanical strength, and barrier effectiveness, while also addressing issues related to tissue engineering scaffold perfusion.

Nano-calcium incorporated piscean collagen scaffolds: potential wound dressing material

BackgroundCollagen proteins extracted from piscean sources are alternatives to bovine and porcine collagen because of their abundance, low price, and skin compatibility and are being explored as suitable wound dressing materials. Intracellular calcium ions are crucial for wound healing, and studies have shown that calcium ion supplementation via an external medium is equally beneficial for speedy recovery. This study explores the wound healing potential of dressing materials that encompass the benefits of nano-calcium and piscean collagen. Nano-calcium sulphate (NCS)-integrated scaffolds were prepared with 100 ppm of NCS and varying concentrations of piscean collagen and HPMC E15 LV. The thickness, tensile strength, folding endurance, pH, expansion profile, and moisture vapour transmission properties of the scaffolds were determined. An in vitro scratch assay and an excision rat wound model were employed to evaluate the wound healing properties of the scaffolds.ResultsThe NCS particles had a mean particle size of 220.7 nm. The scaffolds demonstrated an acceptable thickness, mechanical strength, and flexibility. The scratch assay results revealed that at the end of 24 h of the study, there was an increased wound closure rate with collagen scaffolds in contrast to the control group. In the vivo wound healing studies, formulation CS6 showed 100.0% healing on day 12 as compared to other formulations.ConclusionsWounds treated with scaffolds contracted faster than those treated with a commercial collagen dressing and the control group. The current study thus demonstrates the wound healing ability of nano-calcium sulphate-incorporated piscean collagen scaffolds.

Development of Novel Polysaccharide Membranes for Guided Bone Regeneration: In Vitro and In Vivo Evaluations.

Guided bone regeneration (GBR) procedures require selecting suitable membranes for oral surgery. Pullulan and/or dextran-based polysaccharide materials have shown encouraging results in bone regeneration as bone substitutes but have not been used to produce barrier membranes. The present study aimed to develop and characterize pullulan/dextran-derived membranes for GBR. Two pullulan/dextran-based membranes, containing or not hydroxyapatite (HA) particles, were developed. In vitro, cytotoxicity evaluation was performed using human bone marrow mesenchymal stem cells (hBMSCs). Biocompatibility was assessed on rats in a subcutaneous model for up to 16 weeks. In vivo, rat femoral defects were created on 36 rats to compare the two pullulan/dextran-based membranes with a commercial collagen membrane (Bio-Gide®). Bone repair was assessed radiologically and histologically. Both polysaccharide membranes demonstrated cytocompatibility and biocompatibility. Micro-computed tomography (micro-CT) analyses at two weeks revealed that the HA-containing membrane promoted a significant increase in bone formation compared to Bio-Gide®. At one month, similar effects were observed among the three membranes in terms of bone regeneration. The developed pullulan/dextran-based membranes evidenced biocompatibility without interfering with bone regeneration and maturation. The HA-containing membrane, which facilitated early bone regeneration and offered adequate mechanical support, showed promising potential for GBR procedures.

Collagen-based scaffolds with high wet-state cyclic compressibility for potential oral application

Soft tissue substitutes have been developed to treat gingival recessions to avoid a second surgical site. However, products of pure collagen for clinical application lack their original mechanical strengths and tend to degrade fast in vivo. In this study, a collagen-based scaffold crosslinked with oxidized sodium alginate (OSA-Col) was developed to promote mechanical properties. Compared with commercial products collagen matrix (CM) and collagen sponge (CS), OSA-Col scaffolds presented higher wet-state cyclic compressibility, early anti-degradation ability, similar hemocompatibility and cytocompatibility. Furthermore, in the subcutaneous implantation experiment, OSA2-Col3 scaffolds showed better anti-degradation performance than CS scaffolds and superior neovascularization than CM scaffolds. These results demonstrated that OSA2-Col3 scaffolds had potential as a new soft tissue substitute for the treatment of gingival recessions.

Photochemical Tissue Bonding of Amnion Allograft Membranes for Peripheral Nerve Repair: A Biomechanical Analysis.

Photochemical tissue bonding (PTB) is a technique for peripheral nerve repair in which a collagenous membrane is bonded around approximated nerve ends. Studies using PTB with cryopreserved human amnion have shown promising results in a rat sciatic nerve transection model including a more rapid and complete return of function, larger axon size, and thicker myelination than suture repair. Commercial collagen membranes, such as dehydrated amnion allograft, are readily available, offer ease of storage, and have no risk of disease transmission or tissue rejection. However, the biomechanical properties of these membranes using PTB are currently unknown in comparison to PTB of cryopreserved human amnion and suture neurorrhaphy. Rat sciatic nerves (n = 10 per group) were transected and repaired using either suture neurorrhaphy or PTB with one of the following membranes: cryopreserved human amnion, monolayer human amnion allograft (crosslinked and noncrosslinked), trilayer human amnion/chorion allograft (crosslinked and noncrosslinked), or swine submucosa. Repaired nerves were subjected to mechanical testing. During ultimate stress testing, the repair groups that withstood the greatest strain increases were suture neurorrhaphy (69 ± 14%), PTB with crosslinked trilayer amnion (52 ± 10%), and PTB with cryopreserved human amnion (46 ± 20%), although the differences between these groups were not statistically significant. Neurorrhaphy repairs had a maximum load (0.98 ± 0.30 N) significantly greater than all other repair groups except for noncrosslinked trilayer amnion (0.51 ± 0.27 N). During fatigue testing, all samples repaired with suture, or PTBs with either crosslinked or noncrosslinked trilayer amnion were able to withstand strain increases of at least 50%. PTB repairs with commercial noncrosslinked amnion allograft membranes can withstand physiological strain and have comparable performance to repairs with human amnion, which has demonstrated efficacy in vivo. These results indicate the need for further testing of these membranes using in vivo animal model repairs.

Deepening in the understanding of the role of collagen subunits on the differential molecular arrangement of P. glauca and M. merluccius marine collagens

Decades of extensive efforts on marine collagen extraction and characterization allowed to recognize the unique and excellent characteristics of marine collagen offering advantages over that obtained from terrestrial sources. However, not all marine collagens have the same biochemical characteristics; understanding those at molecular and supramolecular level, is crucial for optimal design of applications. One relevant aspect of collagen characterization is the analysis of its different subunits (α-chains) and their intermolecular cross-links (β- and γ-components), which ultimately determine the specific functions of a particular collagen. Collagens from a teleost and an elasmobranch species were analyzed to understand the influence of their subunit composition and intermolecular crosslinking pattern on their different physicochemical behaviour. For comparative purposes a commercial mammal collagen was included in the study. Although electrophoretic profiles showed the typical composition of type I collagen for hake, blue shark and calf collagen, molar ratios of their α-chains were different indicating a different degree of dimerization of their α2-chains with implications in the presence of a different crosslinking degree pattern. Electrophoresis, amino acid composition, hydrophobicity (RP-HPLC) and molecular weight analysis (GPC-HPLC) results, besides a peptide mapping and an antioxidant activity study of the resultant peptides, would help to understand the role of different subunit collagen composition and different crosslinking pattern in the conformation of a differential quaternary supramolecular structure within different species and its biofunctional implications. The experiments developed would allow to progress in the valorization potential of fish discards and byproducts to explore commercial uses of collagens from marine origin.

Utilization of commercial collagens for preparing well-differentiated human beta cells for confocal microscopy.

With technical advances, confocal and super-resolution microscopy have become powerful tools to dissect cellular pathophysiology. Cell attachment to glass surfaces compatible with advanced imaging is critical prerequisite but remains a considerable challenge for human beta cells. Recently, Phelps et al. reported that human beta cells plated on type IV collagen (Col IV) and cultured in neuronal medium preserve beta cell characteristics. We examined human islet cells plated on two commercial sources of Col IV (C6745 and C5533) and type V collagen (Col V) for differences in cell morphology by confocal microscopy and secretory function by glucose-stimulated insulin secretion (GSIS). Collagens were authenticated by mass spectrometry and fluorescent collagen-binding adhesion protein CNA35. All three preparations allowed attachment of beta cells with high nuclear localization of NKX6.1, indicating a well-differentiated status. All collagen preparations supported robust GSIS. However, the morphology of islet cells differed between the 3 preparations. C5533 showed preferable features as an imaging platform with the greatest cell spread and limited stacking of cells followed by Col V and C6745. A significant difference in attachment behavior of C6745 was attributed to the low collagen contents of this preparation indicating importance of authentication of coating material. Human islet cells plated on C5533 showed dynamic changes in mitochondria and lipid droplets (LDs) in response to an uncoupling agent 2-[2-[4-(trifluoromethoxy)phenyl]hydrazinylidene]-propanedinitrile (FCCP) or high glucose + oleic acid. An authenticated preparation of Col IV provides a simple platform to apply advanced imaging for studies of human islet cell function and morphology.

Investigation of Possible Heavy Metals and Antibiotic Residues in Commercial Collagen

The current study investigated whether commercial collagens were within physiologically acceptable limits to ensure their safer use. For this purpose, 10 of the 25 most popular collagen from fish and farm animals (FA) sold on the internet were randomly selected and purchased from a pharmacy. The zinc, lead, cadmium, mercury, and arsenic levels in these commercial products were then analyzed by ICP-OES. Streptomycin, sulfonamide, tetracycline, and chloramphenicol levels in the samples were determined by HPLC. No lead, mercury or arsenic residues were found in any of the tested samples. The mean cadmium levels in fish and FA collagen samples were not significant between the groups (P=0.2548). The lowest cadmium level in fish collagen samples was 0.152 mg/kg and the highest cadmium level was 0.288 mg/kg. Cadmium levels detected in FA collagen samples ranged from 0.183 mg/kg to 2.78 mg/kg. The mean zinc levels in fish and FA collagen were not significant (P=0.2644). The lowest zinc level in fish collagen was 1.368 mg/kg and the highest was 2673 mg/kg. The lowest and highest zinc levels in FA collagen were 1,750 mg/kg and 1528 mg/kg, respectively. According to the current results, no streptomycin, sulfonamide, and tetracycline residues were found in any of the collagen samples evaluated. Chloramphenicol was only in two fish collagen samples, but these values were below the lower detection limits. The results indicated that the risk of heavy metal and antibiotic residues in commercial collagen sold in our country is low.